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Immunization with the Amastigote Surface Protein-2 (ASP-2) and Trans-sialidase (TS) antigens either in the form of recombinant protein, encoded in plasmids or human adenovirus 5 (hAd5) confers robust protection against various lineages of Trypanosoma cruzi. Herein we generated a chimeric protein containing the most immunogenic regions for T and B cells from TS and ASP-2 (TRASP) and evaluated its immunogenicity in comparison with our standard protocol of heterologous prime-boost using plasmids and hAd5. Mice immunized with TRASP protein associated to Poly-ICLC (Hiltonol) were highly resistant to challenge with T. cruzi, showing a large decrease in tissue parasitism, parasitemia and no lethality. This protection lasted for at least 3 months after the last boost of immunization, being equivalent to the protection induced by DNA/hAd5 protocol. TRASP induced high levels of T. cruzi-specific antibodies and IFNγ-producing T cells and protection was primarily mediated by CD8+ T cells and IFN-γ. We also evaluated the toxicity, immunogenicity, and efficacy of TRASP and DNA/hAd5 formulations in dogs. Mild collateral effects were detected at the site of vaccine inoculation. While the chimeric protein associated with Poly-ICLC induced high levels of antibodies and CD4+ T cell responses, the DNA/hAd5 induced no antibodies, but a strong CD8+ T cell response. Immunization with either vaccine protected dogs against challenge with T. cruzi. Despite the similar efficacy, we conclude that moving ahead with TRASP together with Hiltonol is advantageous over the DNA/hAd5 vaccine due to pre-existing immunity to the adenovirus vector, as well as the cost-benefit for development and large-scale production.

Subclinical (asymptomatic) parasitemia is very common amongstPlasmodium-infected individuals. The immunological mechanisms underlying subclinical parasitemia remain elusive. We investigated the immune regulatory mechanisms behind chronic asymptomaticPlasmodiuminfection using mice lacking humoral immunity (µMT−/−mice). µMT−/−mice became chronically infected, despite lacking outward signs of disease, and exhibited increased macrophage numbers, decreased dendritic and CD4 cells, massive hemozoin accumulation in the spleen and bone marrow, and inadequate hematopoiesis. These changes were accompanied by high circulating levels of interleukin-10 (IL-10), enhanced chromatin accessibility of the STAT3 promoter, and enhanced STAT3 binding to the IL-10 promoter in macrophages. Inhibition of IL-10 signaling, despite promoting parasite clearance, resulted in a proinflammatory response, weight loss, and mortality. These results suggest that epigenetic changes induced by chronicP. chabaudiinfection lead to high levels of circulating IL-10, protecting chronically infected mice against an excessive inflammatory response to high levels of blood-stage parasites.Author summaryMalaria is a life-threatening disease with a range of symptoms, and it is induced in humans by infections with different species ofPlasmodium. Highly prevalent in endemic regions, asymptomaticPlasmodiuminfections are related to long-term exposure to the parasite due to multiple infections and have been demonstrated in human and mouse studies to be associated with elevated levels of IL-10. However, how IL-10 levels remain elevated in the circulation in individuals over the long term has not been determined. We used a mouse model of chronic asymptomaticPlasmodiuminfection to investigate the mechanisms by which IL-10 levels are elevated during chronic asymptomatic infection. Our results show that epigenetic changes in immune genes of myeloid origin could be responsible for the elevated levels of IL-10, and that IL-10 signaling protected chronically infected mice from a severe inflammatory response induced by the infection.

Disorders of carbohydrate metabolism, including hypoglycemia and lactic acidosis, are common features of malaria. In this issue of Cell Metabolism, Ramos et al. report that regulation of gluconeogenesis and glycemia by the host glucose-6-phosphatase catalytic subunit 1 (G6Pc1) is a key metabolic step that affects both Plasmodium replication and clinical outcome of disease.

Both T cells and B cells have been shown to be generated after infection with SARS-CoV-2 yet protocols or experimental models to study one or the other are less common. Here, we generate a chimeric protein (SpiN) that comprises the receptor binding domain (RBD) from Spike (S) and the nucleocapsid (N) antigens from SARS-CoV-2. Memory CD4+ and CD8+ T cells specific for SpiN could be detected in the blood of both individuals vaccinated with Coronavac SARS-CoV-2 vaccine and COVID-19 convalescent donors. In mice, SpiN elicited a strong IFN-γ response by T cells and high levels of antibodies to the inactivated virus, but not detectable neutralizing antibodies (nAbs). Importantly, immunization of Syrian hamsters and the human Angiotensin Convertase Enzyme-2-transgenic (K18-ACE-2) mice with Poly ICLC-adjuvanted SpiN promotes robust resistance to the wild type SARS-CoV-2, as indicated by viral load, lung inflammation, clinical outcome and reduction of lethality. The protection induced by SpiN was ablated by depletion of CD4+ and CD8+ T cells and not transferred by antibodies from vaccinated mice. Finally, vaccination with SpiN also protects the K18-ACE-2 mice against infection with Delta and Omicron SARS-CoV-2 isolates. Hence, vaccine formulations that elicit effector T cells specific for the N and RBD proteins may be used to improve COVID-19 vaccines and potentially circumvent the immune escape by variants of concern.

Funder: Conselho Nacional de Desenvolvimento Científico e Tecnológico, Funder: Fundação de Amparo à Pesquisa do Estado de Minas Gerais, Interleukin-1 receptor-associated kinases (IRAKs) -4, -2, and -1 are involved in transducing signals from Toll-like receptors (TLRs) via the adaptor myeloid differentiation primary-response protein 88 (MYD88). How MYD88/IRAK4/2/1 complexes are formed, their redundancies, and potential non-enzymatic roles are subjects of debate. Here, we examine the hierarchical requirements for IRAK proteins in the context of TLR4 activation and confirmed that the kinase activity of IRAK4 is essential for MYD88 signaling. Surprisingly, the IRAK4 scaffold is required for activation of the E3 ubiquitin ligase TNF receptor-associated factor 6 (TRAF6) by both MYD88 and TIR domain-containing adaptor protein inducing IFN-β (TRIF), a unique adaptation in the TLR4 response. IRAK4 scaffold is, therefore, essential in integrating MYD88 and TRIF in TLR4 signaling.

Activated Vγ9Vδ2 (γδ2) T lymphocytes that sense parasite-produced phosphoantigens are expanded in Plasmodium falciparum–infected patients. Although previous studies suggested that γδ2 T cells help control erythrocytic malaria, whether γδ2 T cells recognize infected red blood cells (iRBCs) was uncertain. Here we show that iRBCs stained for the phosphoantigen sensor butyrophilin 3A1 (BTN3A1). γδ2 T cells formed immune synapses and lysed iRBCs in a contact, phosphoantigen, BTN3A1 and degranulation-dependent manner, killing intracellular parasites. Granulysin released into the synapse lysed iRBCs and delivered death-inducing granzymes to the parasite. All intra-erythrocytic parasites were susceptible, but schizonts were most sensitive. A second protective γδ2 T cell mechanism was identified. In the presence of patient serum, γδ2 T cells phagocytosed and degraded opsonized iRBCs in a CD16-dependent manner, decreasing parasite multiplication. Thus, γδ2 T cells have two ways to control blood-stage malaria–γδ T cell antigen receptor (TCR)-mediated degranulation and phagocytosis of antibody-coated iRBCs. Junqueira et al. show that human γδ T cells control erythrocytic Plasmodium falciparum infection by multiple mechanisms: antibody-dependent phagocytosis, cytotoxic-granule-mediated erythrocyte lysis and direct parasite killing.

After a 2015 outbreak, 23% of patients had retinochoroiditis, indicating that patients with acquired toxoplasmosis should have long-term follow-up, regardless of initial ocular involvement., In 2015, an outbreak of presumed waterborne toxoplasmosis occurred in Gouveia, Brazil. We conducted a 3-year prospective study on a cohort of 52 patients from this outbreak, collected clinical and multimodal imaging findings, and determined risk factors for ocular involvement. At baseline examination, 12 (23%) patients had retinochoroiditis; 4 patients had bilateral and 2 had macular lesions. Multimodal imaging revealed 2 distinct retinochoroiditis patterns: necrotizing focal retinochoroiditis and punctate retinochoroiditis. Older age, worse visual acuity, self-reported recent reduction of visual acuity, and presence of floaters were associated with retinochoroiditis. Among patients, persons >40 years of age had 5 times the risk for ocular involvement. Five patients had recurrences during follow-up, a rate of 22% per person-year. Recurrences were associated with binocular involvement. Two patients had late ocular involvement that occurred >34 months after initial diagnosis. Patients with acquired toxoplasmosis should have long-term ophthalmic follow-up, regardless of initial ocular involvement.

Earlier studies indicate that either the canonical or non-canonical pathways of inflammasome activation have a limited role on malaria pathogenesis. Here, we report that caspase-8 is a central mediator of systemic inflammation, septic shock in the Plasmodium chabaudi-infected mice and the P. berghei-induced experimental cerebral malaria (ECM). Importantly, our results indicate that the combined deficiencies of caspases-8/1/11 or caspase-8/gasdermin-D (GSDM-D) renders mice impaired to produce both TNFα and IL-1β and highly resistant to lethality in these models, disclosing a complementary, but independent role of caspase-8 and caspases-1/11/GSDM-D in the pathogenesis of malaria. Further, we find that monocytes from malaria patients express active caspases-1, -4 and -8 suggesting that these inflammatory caspases may also play a role in the pathogenesis of human disease., Inflammasome activation plays a role in malaria pathogenesis, but details aren’t well understood. Here, the authors show that caspase-8 is a central mediator of systemic inflammation in rodent malaria and that monocytes from malaria patients express active caspases-1, -4 and -8.

Toxoplasmosis is highly endemic worldwide. In Brazil, depending on the geographical region and socioeconomic status, 40–70% of individuals become seropositive at some point in their lives. A significant proportion of Toxoplasma gondii-chronically infected individuals who are otherwise immunocompetent develop recurrent ocular lesions. The inflammatory/immune mechanisms involved in development of ocular lesion are still unknown and, despite previous investigation, there are no reliable immune biomarkers to predict/follow disease outcome. To better understand the impact of the immune response on parasite control and immunopathology of ocular toxoplasmosis, and to provide insights on putative biomarkers for disease monitoring, we assessed the production of a large panel of circulating immune mediators in a longitudinal study of patients with postnatally acquired toxoplasmosis stratified by the presence of ocular involvement, both at the early acute stage and 6 months later during chronic infection, correlating them with presence of ocular involvement. We found that T. gondii-infected patients, especially during the acute stage of the disease, display high levels of chemokines, cytokines, and growth factors involved in the activation, proliferation, and migration of inflammatory cells to injured tissues. In particular, major increases were found in the IFN-induced chemokines CXCL9 and CXCL10 in T. gondii-infected patients regardless of disease stage or clinical manifestations. Moreover, a specific subgroup of circulating cytokines and chemokines including GM-CSF, CCL25, CCL11, CXCL12, CXCL13, and CCL2 was identified as potential biomarkers that accurately distinguish different stages of infection and predict the occurrence of ocular toxoplasmosis. In addition to serving as predictors of disease development, these host inflammatory molecules may offer promise as candidate targets for therapeutic intervention.

Heterogeneity and high plasticity are common features of cells from the mononuclear phagocyte system: monocytes (MOs), macrophages, and dendritic cells (DCs). Upon activation by microbial agents, MO can differentiate into MO-derived DCs (MODCs). In previous work, we have shown that during acute infection with Plasmodium berghei ANKA (PbA), MODCs become, transiently, the main CD11b+ myeloid population in the spleen (SP) and once recruited to the brain play an important role in the development of experimental cerebral malaria (ECM). Here, we isolated 4 cell populations: bone marrow (BM) MOs (BM-MOs) and SP-MOs from uninfected mice; BM inflammatory MOs (BM-iMOs) and SP-MODCs from PbA-infected mice and used a system biology approach to a holistic transcriptomic comparison and provide an interactome analysis by integrating differentially expressed miRNAs (DEMs) and their differentially expressed gene targets (DEGs) data. The Jaccard index (JI) was used for gauging the similarity and diversity among these cell populations. Whereas BM-MOs, BM-iMOs, and SP-MOs presented high similarity of DEGs, SP-MODCs distinguished by showing a greater number of DEGs. Moreover, functional analysis identified an enrichment in canonical pathways, such as DC maturation, neuroinflammation, and IFN signaling. Upstream regulator analysis identified IFNγ as the potential upstream molecule that can explain the observed DEMs–Target DEGs intersections in SP-MODCs. Finally, directed target analysis and in vivo/ex vivo assays indicate that SP-MODCs differentiate in the SP and IFNγ is a main driver of this process.

Trans-sialidases (TS) are unusual enzymes present on the surface of Trypanosoma cruzi, the causative agent of Chagas disease. Encoded by the largest gene family in the T. cruzi genome, only few members of the TS family have catalytic activity. Active trans-sialidases (aTS) are responsible for transferring sialic acid from host glycoconjugates to mucins, also present on the parasite surface. The existence of several copies of TS genes has impaired the use of reverse genetics to study this highly polymorphic gene family. Using CRISPR-Cas9, we generated aTS knockout cell lines displaying undetectable levels of TS activity, as shown by sialylation assays and labeling with antibodies that recognize sialic acid-containing mucins. In vitro infection assays showed that disruption of aTS genes does not affect the parasite’s capacity to invade cells or to escape from the parasitophorous vacuole but resulted in impaired differentiation of amastigotes into trypomastigotes and parasite egress from the cell. When inoculated into mice, aTS mutants were unable to establish infection even in the highly susceptible gamma interferon (IFN-γ) knockout mice. Mice immunized with aTS mutants were fully protected against a challenge infection with the virulent T. cruzi Y strain. Altogether, our results confirmed the role of aTS as a T. cruzi virulence factor and indicated that aTS play a major role during the late stages of intracellular development and parasite egress. Notably, mutants lacking TS activity are completely avirulent in animal models of infection and may be used as a live attenuated vaccine against Chagas disease.

The 1990s saw the rapid reemergence of malaria in Amazonia, where it remains an important public health priority in South America. The Amazonian International Center of Excellence in Malaria Research (ICEMR) was designed to take a multidisciplinary approach toward identifying novel malaria control and elimination strategies. Based on geographically and epidemiologically distinct sites in the Northeastern Peruvian and Western Brazilian Amazon regions, synergistic projects integrate malaria epidemiology, vector biology, and immunology. The Amazonian ICEMR’s overarching goal is to understand how human behavior and other sociodemographic features of human reservoirs of transmission—predominantly asymptomatically parasitemic people—interact with the major Amazonian malaria vector, Nyssorhynchus (formerly Anopheles) darlingi, and with human immune responses to maintain malaria resilience and continued endemicity in a hypoendemic setting. Here, we will review Amazonian ICEMR’s achievements on the synergies among malaria epidemiology, Plasmodium-vector interactions, and immune response, and how those provide a roadmap for further research, and, most importantly, point toward how to achieve malaria control and elimination in the Americas.

The nucleocapsid (N) and the receptor binding domain (RBD) of the Spike (S) proteins elicit robust antibody and T cell responses either in vaccinated or COVID-19 convalescent individuals. We generated a chimeric protein that comprises the sequences of RBD from S and N antigens (SpiN). SpiN was highly immunogenic and elicited a strong IFNγ response from T cells and high levels of antibodies to the inactivated virus, but no neutralizing antibodies (nAb). Importantly, hamsters and the human Angiotensin Convertase Enzyme-2-transgenic mice immunized with SpiN were highly resistant to challenge with the wild type SARS-CoV-2, as indicated by viral load, clinical outcome, lung inflammation and lethality. This protective immunity was dependent of CD4+ T and CD8+ T cells, but not by transfer of antibody of vaccinated mice. Thus, our experiment provides an example T cell-mediated immunity and reinforce the concept that T cell target antigens other that the S protein maybe considered to improve SARS-CoV-2 vaccines, and eventually circumvent the immune scape by variants.

Individuals with asymptomatic infection due to Plasmodium vivax are posited to be important reservoirs of malaria transmission in endemic regions. Here we studied a cohort of P. vivax malaria patients in a suburban area in the Brazilian Amazon. Overall 1,120 individuals were screened for P. vivax infection and 108 (9.6%) had parasitemia detected by qPCR but not by microscopy. Asymptomatic individuals had higher levels of antibodies against P. vivax and similar hematological and biochemical parameters compared to uninfected controls. Blood from asymptomatic individuals with very low parasitemia transmitted P. vivax to the main local vector, Nyssorhynchus darlingi. Lower mosquito infectivity rates were observed when blood from asymptomatic individuals was used in the membrane feeding assay. While blood from symptomatic patients infected 43.4% (199/458) of the mosquitoes, blood from asymptomatic infected 2.5% (43/1,719). However, several asymptomatic individuals maintained parasitemia for several weeks indicating their potential role as an infectious reservoir. These results suggest that asymptomatic individuals are an important source of malaria parasites and Science and Technology for Vaccines granted by Conselho Nacional de may contribute to the transmission of P. vivax in low-endemicity areas of malaria., Author summary Malaria still poses as one of the most important parasitic diseases in the world. The advance of molecular diagnosis brought to light the existence of asymptomatic infections, which may represent most of the infections in some areas. Importantly, the role of asymptomatic carriers in the natural history of malaria is not completely understood. Herein we describe the general characteristics of asymptomatic individuals infected with Plasmodium vivax, and provide evidence of their potential as parasitic reservoirs, even when molecular methods fail to detect the infection. Our findings reinforce the need for better diagnostic tests and open a new window of complexity to be considered in control programs.

Monocytes play an important role in the host defense against Plasmodium vivax as the main source of inflammatory cytokines and mitochondrial reactive oxygen species (mROS). Here, we show that monocyte metabolism is altered during human P. vivax malaria, with mitochondria playing a major function in this switch. The process involves a reprograming in which the cells increase glucose uptake and produce ATP via glycolysis instead of oxidative phosphorylation. P. vivax infection results in dysregulated mitochondrial gene expression and in altered membrane potential leading to mROS increase rather than ATP production. When monocytes were incubated with P. vivax-infected reticulocytes, mitochondria colocalized with phagolysosomes containing parasites representing an important source mROS. Importantly, the mitochondrial enzyme superoxide dismutase 2 (SOD2) is simultaneously induced in monocytes from malaria patients. Taken together, the monocyte metabolic reprograming with an increased mROS production may contribute to protective responses against P. vivax while triggering immunomodulatory mechanisms to circumvent tissue damage. IMPORTANCE Plasmodium vivax is the most widely distributed causative agent of human malaria. To achieve parasite control, the human immune system develops a substantial inflammatory response that is also responsible for the symptoms of the disease. Among the cells involved in this response, monocytes play an important role. Here, we show that monocyte metabolism is altered during malaria, with its mitochondria playing a major function in this switch. This change involves a reprograming process in which the cells increase glucose uptake and produce ATP via glycolysis instead of oxidative phosphorylation. The resulting altered mitochondrial membrane potential leads to an increase in mitochondrial reactive oxygen species rather than ATP. These data suggest that agents that change metabolism should be investigated and used with caution during malaria.

There is a massive demand to identify alternative methods to detect new cases of COVID-19 as well as to investigate the epidemiology of the disease. In many countries, importation of commercial kits poses a significant impact on their testing capacity and increases the costs for the public health system. We have developed an ELISA to detect IgG antibodies against SARS-CoV-2 using a recombinant viral nucleocapsid (rN) protein expressed in E. coli. Using a total of 894 clinical samples we showed that the rN-ELISA was able to detect IgG antibodies against SARS-CoV-2 with high sensitivity (97.5%) and specificity (96.3%) when compared to a commercial antibody test. After three external validation studies, we showed that the test accuracy was higher than 90%. The rN-ELISA IgG kit constitutes a convenient and specific method for the large-scale determination of SARS-Cov-2 antibodies in human sera with high reliability.

Deficiency in memory formation and increased immunosenescence are pivotal features of Trypanosoma cruzi infection proposed to play a role in parasite persistence and disease development. The vaccination protocol that consists in a prime with plasmid DNA followed by the boost with a deficient recombinant human adenovirus type 5, both carrying the ASP2 gene of T. cruzi, is a powerful strategy to elicit effector memory CD8+ T-cells against this parasite. In virus infections, the inhibition of mTOR, a kinase involved in several biological processes, improves the response of memory CD8+ T-cells. Therefore, our aim was to assess the role of rapamycin, the pharmacological inhibitor of mTOR, in CD8+ T response against T. cruzi induced by heterologous prime-boost vaccine. For this purpose, C57BL/6 or A/Sn mice were immunized and daily treated with rapamycin for 34 days. CD8+ T-cells response was evaluated by immunophenotyping, intracellular staining, ELISpot assay and in vivo cytotoxicity. In comparison with vehicle-injection, rapamycin administration during immunization enhanced the frequency of ASP2-specific CD8+ T-cells and the percentage of the polyfunctional population, which degranulated (CD107a+) and secreted both interferon gamma (IFNγ) and tumor necrosis factor (TNF). The beneficial effects were long-lasting and could be detected 95 days after priming. Moreover, the effects were detected in mice immunized with ten-fold lower doses of plasmid/adenovirus. Additionally, the highly susceptible to T. cruzi infection A/Sn mice, when immunized with low vaccine doses, treated with rapamycin, and challenged with trypomastigote forms of the Y strain showed a survival rate of 100%, compared with 42% in vehicle-injected group. Trying to shed light on the biological mechanisms involved in these beneficial effects on CD8+ T-cells by mTOR inhibition after immunization, we showed that in vivo proliferation was higher after rapamycin treatment compared with vehicle-injected group. Taken together, our data provide a new approach to vaccine development against intracellular parasites, placing the mTOR inhibitor rapamycin as an adjuvant to improve effective CD8+ T-cell response.

Summary. Human visceral leishmaniasis (VL) is a major public health problem worldwide, leading to significant mortality rates if not properly treated and controlled. Precise identification of infected patients is essential to establish treatment and control measures. Although several VL serological diagnosis advances have been accomplished lately, mainly using recombinant antigens and immunochromatographic tests (ICTs), improvements may still be achieved using multiepitope chimeric proteins in different test platforms. Here, we reported on the evaluation of ELISA and an ICT developed with a new chimeric protein, named DTL-4, based on repetitive antigenic sequences, including those present in the A2 protein. Methods. A total of 1028 sera samples were used for the development and validation of ELISA (321 samples from L. infantum-infected patients, 62 samples from VL/AIDS coinfected patients, 236 samples from patients infected with other diseases, and 409 samples from healthy donors). A total of 520 sera samples were used to develop and validate ICT (249 samples from L. infantum-infected patients, 46 samples from VL/AIDS coinfected patients, 40 samples from patients infected with other diseases, and 185 samples from healthy donors). Findings. Using the validation sera panels, DTL-4-based ELISA displayed an overall sensitivity of 94.61% (95% CI: 89.94-97.28), a specificity of 99.41% (95% CI: 96.39-99.99), and an accuracy of 97.02% (95% CI: 94.61-98.38), while for ICT, sensitivity, specificity, and accuracy values corresponded to 91.98% (95% CI: 86.65-95.39), 100.00% (95% CI: 96.30-100.00), and 95.14% (95% CI: 91.62-97.15), respectively. When testing sera samples from VL/AIDS coinfected patients, DTL-4-ELISA displayed a sensitivity of 77.42% (95% CI: 65.48-86.16), a specificity of 99.41% (95% CI: 96.39-99.99), and an accuracy of 93.51% (95% CI: 89.49%-96.10%), while for DTL-4-ICT, sensitivity was 73.91% (95% CI: 59.74-84.40), specificity was 90.63% (95% CI: 81.02-95.63), and accuracy was 82.00% (95% CI: 73.63-90.91). Conclusion. DTL-4 is a promising candidate antigen for serodiagnosis of VL patients, including those with VL/AIDS coinfection, when incorporated into ELISA or ICT test formats.

There is a massive demand to identify alternative methods to detect new cases of COVID-19 as well as to investigate the epidemiology of the disease. In many countries, importation of commercial kits poses a significant impact on their testing capacity and increases the costs for the public health system. We have developed an ELISA to detect IgG antibodies against SARS-CoV-2 using a recombinant viral nucleocapsid (rN) protein expressed in

Human primary monocytes are composed of a minor, more mature CD16+(CD14low/neg) population and a major CD16neg(CD14+) subset. The specific functions of CD16+ versus CD16neg monocytes in steady state or inflammation remain poorly understood. In previous work, we found that IL-12 is selectively produced by the CD16+ subset in response to the protozoan pathogen, Toxoplasma gondii. In this study, we demonstrated that this differential responsiveness correlates with the presence of an IFN-induced transcriptional signature in CD16+ monocytes already at baseline. Consistent with this observation, we found that in vitro IFN-γ priming overcomes the defect in the IL-12 response of the CD16neg subset. In contrast, pretreatment with IFN-γ had only a minor effect on IL-12p40 secretion by the CD16+ population. Moreover, inhibition of the mTOR pathway also selectively increased the IL-12 response in CD16neg but not in CD16+ monocytes. We further demonstrate that in contrast to IFN-γ, IFN-α fails to promote IL-12 production by the CD16neg subset and blocks the effect of IFN-γ priming. Based on these observations, we propose that the acquisition of IL-12 responsiveness by peripheral blood monocyte subsets depends on extrinsic signals experienced during their developmental progression in vivo. This process can be overridden during inflammation by the opposing regulatory effects of type I and II IFN as well as the mTOR inhibition.

The nucleocapsid (N) and the receptor binding domain (RBD) of the Spike (S) proteins elicit robust antibody and T cell responses either in vaccinated or COVID-19 convalescent individuals. We generated a chimeric protein that comprises the sequences of RBD from S and N antigens (SpiN). SpiN was highly immunogenic and elicited a strong IFNγ response from T cells and high levels of antibodies to the inactivated virus, but no neutralizing antibodies. Importantly, hamsters and the human Angiotensin Convertase Enzyme-2-transgenic mice immunized with SpiN were highly resistant to challenge with the wild type SARS-CoV-2, as indicated by viral load, clinical outcome, lung inflammation and lethality. Thus, the N protein should be considered to induce T-cell-based immunity to improve SARS-CoV-2 vaccines, and eventually to circumvent the immune scape by variants.

Leishmania parasites use elaborate virulence mechanisms to invade and thrive in macrophages. These virulence mechanisms inhibit host cell defense responses and generate a specialized replicative niche, the parasitophorous vacuole. In this work, we performed a genome-wide RNAi screen in Drosophila macrophage-like cells to identify host factors necessary for Leishmania amazonensis infection. This screen identified 52 conserved genes required specifically for parasite entry, including several components of the SUMOylation machinery. Further studies in mammalian macrophages found that L. amazonensis infection inhibited SUMOylation within infected macrophages and this inhibition enhanced parasitophorous vacuole growth and parasite proliferation through modulation of multiple genes especially ATP6V0D2, which in turn effects CD36 expression and cholesterol levels. Together, these data suggest that parasites actively sabotage host SUMOylation and alter host transcription to improve their intracellular niche and enhance their replication.

Toxoplasmosis affects one-third of the human population worldwide. Humans are accidental hosts and are infected after consumption of undercooked meat and water contaminated with Toxoplasma gondii cysts and oocysts, respectively. Neutrophils have been shown to participate in the control of T. gondii infection in mice through a variety of effector mechanisms, such as reactive oxygen species (ROS) and neutrophil extracellular trap (NET) formation. However, few studies have demonstrated the role of neutrophils in individuals naturally infected with T. gondii. In the current study, we evaluated the activation status of neutrophils in individuals with acute or chronic toxoplasmosis and determined the role of T. gondii-induced NET formation in the amplification of the innate and adaptive immune responses. We observed that neutrophils are highly activated during acute infection through increased expression of CD66b. Moreover, neutrophils from healthy donors (HDs) cocultured with tachyzoites produced ROS and formed NETs, with the latter being dependent on glycolysis, succinate dehydrogenase, gasdermin D, and neutrophil elastase. Furthermore, we observed elevated levels of the chemokines (CXC motif) CXCL8 and (CC motif) CCL4 ligands in plasma from patients with acute toxoplasmosis and production by neutrophils from HDs exposed to T. gondii. Finally, we showed that T. gondii-induced NETs activate neutrophils and promote the recruitment of autologous CD4+ T cells and the production of interferon gamma (IFN-γ), tumor necrosis factor (TNF), interleukin 6 (IL-6), IL-17, and IL-10 by peripheral blood mononuclear cells. In conclusion, we demonstrated that T. gondii activates neutrophils and promotes the release of NETs, which amplify human innate and adaptive immune responses. IMPORTANCE Approximately one-third of the human population is estimated to be chronically infected with the obligate intracellular parasite Toxoplasma gondii. Humans are accidental hosts that are infected with T. gondii after consumption of undercooked meat or contaminated water. Neutrophils have been shown to control T. gondii growth by different mechanisms, including neutrophil extracellular traps (NETs). In the current study, we observed that neutrophils are highly activated during acute toxoplasmosis. We also determined that T. gondii-induced NETs are dependent on the energetic profile of neutrophils as well as the production of ROS and gasdermin D (GSDMD) cleavage. In addition, we showed that T. gondii-induced NETs activate neutrophils, promote the recruitment of autologous CD4+ T cells, and induce the production of cytokines by peripheral blood mononuclear cells, amplifying the innate and adaptive immune responses.

The COVID-19 pandemic is shining a spotlight on the field of immunology like never before. To appreciate the diverse ways in which immunologists have contributed, Nature Reviews Immunology invited the president of the International Union of Immunological Societies and the presidents of 15 other national immunology societies to discuss how they and their members responded following the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)., For this Viewpoint, Nature Reviews Immunology asked the presidents of 16 immunology societies from around the world to discuss how their society and its members responded to the COVID-19 pandemic. Their answers highlight the incredible contributions that immunologists around the globe have made following the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

All authors: Andrea Cossarizza Hyun‐Dong Chang Andreas Radbruch Andreas Acs Dieter Adam Sabine Adam‐Klages William W. Agace Nima Aghaeepour Mübeccel Akdis Matthieu Allez Larissa Nogueira Almeida Giorgia Alvisi Graham Anderson Immanuel Andrä Francesco Annunziato Achille Anselmo Petra Bacher Cosima T. Baldari Sudipto Bari Vincenzo Barnaba Joana Barros‐Martins Luca Battistini Wolfgang Bauer Sabine Baumgart Nicole Baumgarth Dirk Baumjohann Bianka Baying Mary Bebawy Burkhard Becher Wolfgang Beisker Vladimir Benes Rudi Beyaert Alfonso Blanco Dominic A. Boardman Christian Bogdan Jessica G. Borger Giovanna Borsellino Philip E. Boulais Jolene A. Bradford Dirk Brenner Ryan R. Brinkman Anna E. S. Brooks Dirk H. Busch Martin Büscher Timothy P. Bushnell Federica Calzetti Garth Cameron Ilenia Cammarata Xuetao Cao Susanna L. Cardell Stefano Casola Marco A. Cassatella Andrea Cavani Antonio Celada Lucienne Chatenoud Pratip K. Chattopadhyay Sue Chow Eleni Christakou Luka Čičin‐Šain Mario Clerici Federico S. Colombo Laura Cook Anne Cooke Andrea M. Cooper Alexandra J. Corbett Antonio Cosma Lorenzo Cosmi Pierre G. Coulie Ana Cumano Ljiljana Cvetkovic Van Duc Dang Chantip Dang‐Heine Martin S. Davey Derek Davies Sara De Biasi Genny Del Zotto Gelo Victoriano Dela Cruz Michael Delacher Silvia Della Bella Paolo Dellabona Günnur Deniz Mark Dessing James P. Di Santo Andreas Diefenbach Francesco Dieli Andreas Dolf Thomas Dörner Regine J. Dress Diana Dudziak Michael Dustin Charles‐Antoine Dutertre Friederike Ebner Sidonia B. G. Eckle Matthias Edinger Pascale Eede Götz R.A. Ehrhardt Marcus Eich Pablo Engel Britta Engelhardt Anna Erdei Charlotte Esser Bart Everts Maximilien Evrard Christine S. Falk Todd A. Fehniger Mar Felipo‐Benavent Helen Ferry Markus Feuerer Andrew Filby Kata Filkor Simon Fillatreau Marie Follo Irmgard Förster John Foster Gemma A. Foulds Britta Frehse Paul S. Frenette Stefan Frischbutter Wolfgang Fritzsche David W. Galbraith Anastasia Gangaev Natalio Garbi Brice Gaudilliere Ricardo T. Gazzinelli Jens Geginat Wilhelm Gerner Nicholas A. Gherardin Kamran Ghoreschi Lara Gibellini Florent Ginhoux Keisuke Goda Dale I. Godfrey Christoph Goettlinger Jose M. González‐Navajas Carl S. Goodyear Andrea Gori Jane L. Grogan Daryl Grummitt Andreas Grützkau Claudia Haftmann Jonas Hahn Hamida Hammad Günter Hämmerling Leo Hansmann Goran Hansson Christopher M. Harpur Susanne Hartmann Andrea Hauser Anja E. Hauser David L. Haviland David Hedley Daniela C. Hernández Guadalupe Herrera Martin Herrmann Christoph Hess Thomas Höfer Petra Hoffmann Kristin Hogquist Tristan Holland Thomas Höllt Rikard Holmdahl Pleun Hombrink Jessica P. Houston Bimba F. Hoyer Bo Huang Fang‐Ping Huang Johanna E. Huber Jochen Huehn Michael Hundemer Christopher A. Hunter William Y. K. Hwang Anna Iannone Florian Ingelfinger Sabine M Ivison Hans‐Martin Jäck Peter K. Jani Beatriz Jávega Stipan Jonjic Toralf Kaiser Tomas Kalina Thomas Kamradt Stefan H. E. Kaufmann Baerbel Keller Steven L. C. Ketelaars Ahad Khalilnezhad Srijit Khan Jan Kisielow Paul Klenerman Jasmin Knopf Hui‐Fern Koay Katja Kobow Jay K. Kolls Wan Ting Kong Manfred Kopf Thomas Korn Katharina Kriegsmann Hendy Kristyanto Thomas Kroneis Andreas Krueger Jenny Kühne Christian Kukat Désirée Kunkel Heike Kunze‐Schumacher Tomohiro Kurosaki Christian Kurts Pia Kvistborg Immanuel Kwok Jonathan Landry Olivier Lantz Paola Lanuti Francesca LaRosa Agnès Lehuen Salomé LeibundGut‐Landmann Michael D. Leipold Leslie Y.T. Leung Megan K. Levings Andreia C. Lino Francesco Liotta Virginia Litwin Yanling Liu Hans‐Gustaf Ljunggren Michael Lohoff Giovanna Lombardi Lilly Lopez Miguel López‐Botet Amy E. Lovett‐Racke Erik Lubberts Herve Luche Burkhard Ludewig Enrico Lugli Sebastian Lunemann Holden T. Maecker Laura Maggi Orla Maguire Florian Mair Kerstin H. Mair Alberto Mantovani Rudolf A. Manz Aaron J. Marshall Alicia Martínez‐Romero Glòria Martrus Ivana Marventano Wlodzimierz Maslinski Giuseppe Matarese Anna Vittoria Mattioli Christian Maueröder Alessio Mazzoni James McCluskey Mairi McGrath Helen M. McGuire Iain B. McInnes Henrik E. Mei Fritz Melchers Susanne Melzer Dirk Mielenz Stephen D. Miller Kingston H.G. Mills Hans Minderman Jenny Mjösberg Jonni Moore Barry Moran Lorenzo Moretta Tim R. Mosmann Susann Müller Gabriele Multhoff Luis Enrique Muñoz Christian Münz Toshinori Nakayama Milena Nasi Katrin Neumann Lai Guan Ng Antonia Niedobitek Sussan Nourshargh Gabriel Núñez José‐Enrique O'Connor Aaron Ochel Anna Oja Diana Ordonez Alberto Orfao Eva Orlowski‐Oliver Wenjun Ouyang Annette Oxenius Raghavendra Palankar Isabel Panse Kovit Pattanapanyasat Malte Paulsen Dinko Pavlinic Livius Penter Pärt Peterson Christian Peth Jordi Petriz Federica Piancone Winfried F. Pickl Silvia Piconese Marcello Pinti A. Graham Pockley Malgorzata Justyna Podolska Zhiyong Poon Katharina Pracht Immo Prinz Carlo E. M. Pucillo Sally A. Quataert Linda Quatrini Kylie M. Quinn Helena Radbruch Tim R. D. J. Radstake Susann Rahmig Hans‐Peter Rahn Bartek Rajwa Gevitha Ravichandran Yotam Raz Jonathan A. Rebhahn Diether Recktenwald Dorothea Reimer Caetano Reis e Sousa Ester B.M. Remmerswaal Lisa Richter Laura G. Rico Andy Riddell Aja M. Rieger J. Paul Robinson Chiara Romagnani Anna Rubartelli Jürgen Ruland Armin Saalmüller Yvan Saeys Takashi Saito Shimon Sakaguchi Francisco Sala‐de‐Oyanguren Yvonne Samstag Sharon Sanderson Inga Sandrock Angela Santoni Ramon Bellmàs Sanz Marina Saresella Catherine Sautes‐Fridman Birgit Sawitzki Linda Schadt Alexander Scheffold Hans U. Scherer Matthias Schiemann Frank A. Schildberg Esther Schimisky Andreas Schlitzer Josephine Schlosser Stephan Schmid Steffen Schmitt Kilian Schober Daniel Schraivogel Wolfgang Schuh Thomas Schüler Reiner Schulte Axel Ronald Schulz Sebastian R. Schulz Cristiano Scottá Daniel Scott‐Algara David P. Sester T. Vincent Shankey Bruno Silva‐Santos Anna Katharina Simon Katarzyna M. Sitnik Silvano Sozzani Daniel E. Speiser Josef Spidlen Anders Stahlberg Alan M. Stall Natalie Stanley Regina Stark Christina Stehle Tobit Steinmetz Hannes Stockinger Yousuke Takahama Kiyoshi Takeda Leonard Tan Attila Tárnok Gisa Tiegs Gergely Toldi Julia Tornack Elisabetta Traggiai Mohamed Trebak Timothy I.M. Tree Joe Trotter John Trowsdale Maria Tsoumakidou Henning Ulrich Sophia Urbanczyk Willem van de Veen Maries van den Broek Edwin van der Pol Sofie Van Gassen Gert Van Isterdael René A.W. van Lier Marc Veldhoen Salvador Vento‐Asturias Paulo Vieira David Voehringer Hans‐Dieter Volk Anouk von Borstel Konrad von Volkmann Ari Waisman Rachael V. Walker Paul K. Wallace Sa A. Wang Xin M. Wang Michael D. Ward Kirsten A Ward‐Hartstonge Klaus Warnatz Gary Warnes Sarah Warth Claudia Waskow James V. Watson Carsten Watzl Leonie Wegener Thomas Weisenburger Annika Wiedemann Jürgen Wienands Anneke Wilharm Robert John Wilkinson Gerald Willimsky James B. Wing Rieke Winkelmann Thomas H. Winkler Oliver F. Wirz Alicia Wong Peter Wurst Jennie H. M. Yang Juhao Yang Maria Yazdanbakhsh Liping Yu Alice Yue Hanlin Zhang Yi Zhao Susanne Maria Ziegler Christina Zielinski Jakob Zimmermann Arturo Zychlinsky., These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion., This work was supported by the Netherlands Organisation for Scientific Research – Domain Applied and Engineering Sciences (NWO-TTW), research program VENI 15924. This work was funded by the Deutsche Forschungsgemeinschaft. European Union Innovative Medicines Initiative - Joint Undertaking - RTCure Grant Agreement 777357 and innovation program (Grant Agreement 695551).

This study investigated the efficacy of the vaccine in liver of mice infected with the Trypanosoma cruzi ( T. cruzi ) and immunized with AdASP-2. For this purpose, histopathological analysis and gene expression of COX-2, TNF-alpha, TNFR, iNOS, cytochrome C , caspase-3, TLR4, IL-6 and IL10 were evaluated. The following groups were used in this study: Group 1 - Control Group (CTRL) animals received AdβGal vehicle; Group 2 - Infected Group (TC) animals were infected with T. cruzi ; Group 3 - Immunized Group (AdASP-2): animals were immunized by AdASP-2 vaccine; Group 4 - Immunized and Infected Group (AdASP-2+TC) animals were infected with T. cruzi and immunized by AdSP-2 vaccine. A significant decrease of amastigote nests was noticed in the group of animals that were immunized with AdASP-2 and infected on the same day. COX-2 and TNF-alpha gene expressions increased in TC group, whereas TNF-alpha decreased in the TC+AdASP-2 group. TNFR expression was high in AdASP-2+TC group. iNOS expression was high for all experimental groups whereas cytochrome C decreased for all experimental groups. Caspase 3 increased in TC and TC+AdASP-2 groups. The gene expression of TLR4 and IL-10 showed an increase in AdASP-2+TC group. Finally, hepatic fibrosis was noticed to TC and AdASP-2 + TC groups. Taken together, our results demonstrated that vaccination with AdASP-2 was effective against the acute phase of experimental Chagas disease as a result of a more powerful and rapid immune response closely related to expression of some inflammatory genes, such as iNOS, TNF-alpha, TLR 4, and IL-10.

Previous studies have indicated that antibody responses can be robustly induced after the vaccination in individuals previously infected by SARS-CoV-2. To evaluate anti-SARS-CoV-2 humoral responses in vaccinated individuals with or without a previous history of COVID-19, we compared levels of anti-SARS-CoV-2 antibodies in the sera from 21 vaccinees, including COVID-19-recovered or -naïve individuals in different times, before and after immunization with an inactivated COVID-19 vaccine. Anti-SARS-CoV-2-specific antibodies elicited after COVID-19 and/or immunization with an inactivated vaccine were measured by ELISA and Plaque Reduction Neutralizing assays. Antibody kinetics were consistently different between the two vaccine doses for naïve individuals, contrasting with the SARS-CoV-2-recovered subjects in which we observed no additional increase in antibody levels following the second dose. Sera from SARS-CoV2-naïve individuals had no detectable neutralizing activity against lineage B.1 SARS-CoV-2 or Gamma variant five months after the second vaccine dose. Contrarily, SARS-CoV-2-recovered subjects retained considerable neutralizing activity against both viruses. We conclude that a single inactivated SARS-CoV-2 vaccine dose may be sufficient to induce protective antibody responses in individuals with previous history of SARS-CoV-2 infection.

Malaria-associated acute respiratory distress syndrome (MA-ARDS) and acute lung injury (ALI) are complications that cause lung damage and often leads to death. The MA-ARDS/ALI is associated with a Type 1 inflammatory response mediated by T lymphocytes and IFN-γ. Here, we used the Plasmodium berghei NK65 (PbN)-induced MA-ALI/ARDS model that resembles human disease and confirmed that lung CD4+ and CD8+ T cells predominantly expressed Tbet and IFN-γ. Surprisingly, we found that development of MA-ALI/ARDS was dependent on functional CCR4, known to mediate the recruitment of Th2 lymphocytes and regulatory T cells. However, in this Type 1 inflammation-ARDS model, CCR4 was not involved in the recruitment of T lymphocytes, but was required for the emergence of TNF-α/iNOS producing dendritic cells (Tip-DCs) in the lungs. In contrast, recruitment of Tip-DCs and development of MA-ALI/ARDS were not altered in CCR2−/− mice. Importantly, we showed that NOS2−/− mice are resistant to PbN-induced lung damage, indicating that reactive nitrogen species produced by Tip-DCs play an essential role in inducing MA-ARDS/ALI. Lastly, our experiments suggest that production of IFN-γ primarily by CD8+ T cells is required for inducing Tip-DCs differentiation in the lungs and the development of MA-ALI/ARDS model. A síndrome do desconforto respiratório agudo associado à malária (MA-ARDS) e a lesão pulmonar aguda (LPA) são complicações que causam danos nos pulmões e muitas vezes levam à morte. A MA-ARDS/ALI está associada a uma resposta inflamatória tipo 1 mediada por linfócitos T e IFN-γ. Aqui, usamos o modelo MA-ALI/ARDS induzido por Plasmodium berghei NK65 (PbN) que se assemelha à doença humana e confirmamos que as células T CD4+ e CD8+ do pulmão expressavam predominantemente Tbet e IFN-γ. Surpreendentemente, descobrimos que o desenvolvimento de MA-ALI/ARDS era dependente de CCR4 funcional, conhecido por mediar o recrutamento de linfócitos Th2 e células T reguladoras. No entanto, neste modelo de inflamação-ARDS Tipo 1, o CCR4 não estava envolvido no recrutamento de linfócitos T, mas era necessário para o surgimento de células dendríticas produtoras de TNF-α/iNOS (Tip-DCs) nos pulmões. Em contraste, o recrutamento de Tip-DCs e o desenvolvimento de MA-ALI/ARDS não foram alterados em camundongos CCR2-/-. É importante ressaltar que mostramos que camundongos NOS2-/- são resistentes a danos pulmonares induzidos por PbN, indicando que espécies reativas de nitrogênio produzidas por Tip-DCs desempenham um papel essencial na indução de MA-ARDS/ALI. Por fim, nossos experimentos sugerem que a produção de IFN-γ principalmente por células T CD8+ é necessária para induzir a diferenciação de Tip-DCs nos pulmões e o desenvolvimento do modelo MA-ALI/ARDS.

Plasmodium vivax causes approximately 100 million clinical malaria cases yearly1,2. The basis of protective immunity is poorly understood and thought to be mediated by antibodies3,4. Cytotoxic CD8+ T cells protect against other intracellular parasites by detecting parasite peptides presented by human leukocyte antigen class I on host cells. Cytotoxic CD8+ T cells kill parasite-infected mammalian cells and intracellular parasites by releasing their cytotoxic granules5,6. Perforin delivers the antimicrobial peptide granulysin and death-inducing granzymes into the host cell, and granulysin then delivers granzymes into the parasite. Cytotoxic CD8+ T cells were thought to have no role against Plasmodium spp. blood stages because red blood cells generally do not express human leukocyte antigen class I7. However, P. vivax infects reticulocytes that retain the protein translation machinery. Here we show that P. vivax–infected reticulocytes express human leukocyte antigen class I. Infected patient circulating CD8+ T cells highly express cytotoxic proteins and recognize and form immunological synapses with P. vivax–infected reticulocytes in a human leukocyte antigen–dependent manner, releasing their cytotoxic granules to kill both host cell and intracellular parasite, preventing reinvasion. P. vivax–infected reticulocytes and parasite killing is perforin independent, but depends on granulysin, which generally efficiently forms pores only in microbial membranes8. We find that P. vivax depletes cholesterol from the P. vivax–infected reticulocyte cell membrane, rendering it granulysin-susceptible. This unexpected T cell defense might be mobilized to improve P. vivax vaccine efficacy. T cells kill blood-stage Plasmodium vivax, providing a rationale for the development of a T cell vaccine against this parasite.

The balance between pro- and antiinflammatory mechanisms is essential to limit immune-mediated pathology, and CD4(+) forkhead box P3 (Foxp3(+)) regulatory T cells (Treg) play an important role in this process. The expression of inhibitory receptors regulates cytokine production by Plasmodium vivax-specific T cells. Our goal was to assess the induction of programmed death-1 (PD-1) and cytotoxic T-lymphocyte antigen (CTLA-4) on Treg during malaria and to evaluate their function. We found that P. vivax infection triggered an increase in circulating Treg and their expression of CTLA-4 and PD-1. Functional analysis demonstrated that Treg from malaria patients had impaired suppressive ability and PD-1(+)Treg displayed lower levels of Foxp3 and Helios, but had higher frequencies of T-box transcription factor(+) and interferon-gamma(+) cells than PD-1(−)Treg. Thus malaria infection alters the function of circulating Treg by triggering increased expression of PD-1 on Treg that is associated with decreased regulatory function and increased proinflammatory characteristics.

The Apicomplexa protozoanToxoplasma gondiiis a mandatory intracellular parasite and the causative agent of toxoplasmosis. This illness is of medical importance due to its high prevalence worldwide and may cause neurological alterations in immunocompromised persons. In chronically infected immunocompetent individuals, this parasite forms tissue cysts mainly in the brain. In addition,T.gondiiinfection has been related to mental illnesses such as schizophrenia, bipolar disorder, depression, obsessive-compulsive disorder, as well as mood, personality, and other behavioral changes. In the present study, we evaluated the kinetics of behavioral alterations in a model of chronic infection, assessing anxiety, depression and exploratory behavior, and their relationship with neuroinflammation and parasite cysts in brain tissue areas, blood-brain-barrier (BBB) integrity, and cytokine status in the brain and serum. Adult female C57BL/6 mice were infected by gavage with 5 cysts of the ME-49 type IIT.gondiistrain, and analyzed as independent groups at 30, 60 and 90 days postinfection (dpi). Anxiety, depressive-like behavior, and hyperactivity were detected in the early (30 dpi) and long-term (60 and 90 dpi) chronicT.gondiiinfection, in a direct association with the presence of parasite cysts and neuroinflammation, independently of the brain tissue areas, and linked to BBB disruption. These behavioral alterations paralleled the upregulation of expression of tumor necrosis factor (TNF) and CC-chemokines (CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β and CCL5/RANTES) in the brain tissue. In addition, increased levels of interferon-gamma (IFNγ), TNF and CCL2/MCP-1 were detected in the peripheral blood, at 30 and 60 dpi. Our data suggest that the persistence of parasite cysts induces sustained neuroinflammation, and BBB disruption, thus allowing leakage of cytokines of circulating plasma into the brain tissue. Therefore, all these factors may contribute to behavioral changes (anxiety, depressive-like behavior, and hyperactivity) in chronicT.gondiiinfection.

Visceral leishmaniasis (VL) is a neglected tropical disease with dogs serving as reservoirs for one of its etiological agents, Leishmania infantum. In Brazil, VL control involves culling of seropositive dogs, among other actions. However, the most employed serological tests lack accuracy, and are not able to detect canine visceral leishmaniasis (CVL) during the early stages of infection. Early detection of CVL is highly desirable in order to shorten the contact time between the infected reservoirs and the vectors. In this study, we investigated the ability of two multiepitope proteins, PQ10 and PQ20, to detect CVL at earlier stages than currently employed methods, including ITS-1 conventional PCR. Using serum samples from naturally infected dogs, we observed that ELISA-PQ10 and ELISA-PQ20 were able to detect Leishmania infection at earlier time points as compared with kDNA PCR-RFLP in anti-IgG and anti-IgM assays. Using sera from experimentally infected dogs, we monitored seroconversion using multiepitope proteins, ELISA-crude antigen, as well as ITS-1 conventional and real-time PCR. While seroconversion was detected by ELISA-crude antigen in 16.6% of the dogs, multiepitope proteins were able to detect seroconversion in more than 80% of them. Moreover, the ability of ELISA-PQ10 and ELISA-PQ20 to detect Leishmania infection at earlier time points as compared with conventional PCR was also confirmed in experimental infection dogs' sera. Immunofluorescence to Babesia canis and Ehrlichia canis did not show cross-reactions with ELISA-PQ10/PQ20 positive samples. Results of real-time PCR and ELISA with multiepitope proteins were very similar, with concordances between 80 and 100%. Furthermore, our findings indicated that PQ10 and PQ20 immunoassays can be related to parasite load. ELISA-PQ10 and ELISA-PQ20 are more sensitive diagnostic tools for early CVL detection as compared with other methods They could potentially be used in screening tests due to easy execution and low costs facilities.

Secreted and surface-displayed carbohydrates are essential for virulence and viability of many parasites, including for immune system evasion. We have identified the α-Gal trisaccharide epitope on the surface of the protozoan parasites Leishmania infantum and Leishmania amazonensis, the etiological agents of visceral and cutaneous leishmaniasis, respectively, with the latter bearing larger amounts of α-Gal than the former. A polyvalent α-Gal conjugate on the immunogenic Qβ virus-like particle was tested as a vaccine against Leishmania infection in a C57BL/6 α-galactosyltransferase knockout mouse model, which mimics human hosts in producing high titers of anti-α-Gal antibodies. As expected, α-Gal-T knockout mice infected with promastigotes of both Leishmania species showed significantly lower parasite load in the liver and slightly decreased levels in the spleen, compared with wild-type mice. Vaccination with Qβ–α-Gal nanoparticles protected the knockout mice against Leishmania challenge, eliminating the i...

Biological rhythms appear to be an elegant solution to the challenge of coordinating activities with the consequences of the Earth's daily and seasonal rotation. The genes and molecular mechanisms underpinning circadian clocks in multicellular organisms are well understood. In contrast, the regulatory mechanisms and fitness consequences of biological rhythms exhibited by parasites remain mysterious. Here, we explore how periodicity in parasite traits is generated and why daily rhythms matter for parasite fitness. We focus on malaria (Plasmodium) parasites which exhibit developmental rhythms during replication in the mammalian host's blood and in transmission to vectors. Rhythmic in-host parasite replication is responsible for eliciting inflammatory responses, the severity of disease symptoms, and fueling transmission, as well as conferring tolerance to anti-parasite drugs. Thus, understanding both how and why the timing and synchrony of parasites are connected to the daily rhythms of hosts and vectors may make treatment more effective and less toxic to hosts.

Chemokine receptor type 3 (CXCR3) plays an important role in CD8+ T cells migration during intracellular infections, such as Trypanosoma cruzi. In addition to chemotaxis, CXCR3 receptor has been described as important to the interaction between antigen-presenting cells and effector cells. We hypothesized that CXCR3 is fundamental to T. cruzi-specific CD8+ T cell activation, migration and effector function. Anti-CXCR3 neutralizing antibody administration to acutely T. cruzi-infected mice decreased the number of specific CD8+ T cells in the spleen, and those cells had impaired in activation and cytokine production but unaltered proliferative response. In addition, anti-CXCR3-treated mice showed decreased frequency of CD8+ T cells in the heart and numbers of plasmacytoid dendritic cells in spleen and lymph node. As CD8+ T cells interacted with plasmacytoid dendritic cells during infection by T. cruzi, we suggest that anti-CXCR3 treatment lowers the quantity of plasmacytoid dendritic cells, which may contribute to impair the prime of CD8+ T cells. Understanding which molecules and mechanisms guide CD8+ T cell activation and migration might be a key to vaccine development against Chagas disease as those cells play an important role in T. cruzi infection control., Author summary Inflammatory chemokine receptors such as CXCR3 play an important role in T lymphocytes migration into an infected tissue during Th1 response. Recently, the role of CXCR3 as a co-stimulatory molecule was demonstrated, and T lymphocytes from CXCR3 deficient mice had impaired effector function. CXCR3 receptor was highly expressed on specific CD8+ T cells after challenge with T. cruzi, and the hypothesis of that molecule is important for CD8+ T cells activation, migration and functionality was raised. We used the anti-CXCR3 neutralizing antibody approach and demonstrated that C57BL/6 treated mice died very quickly due to T. cruzi infection, and specific CD8+ T cells had decreased effector phenotyping, cytokine production, and cytotoxicity. In addition, anti-CXCR3 treatment decreased the number of dendritic plasmacytoid cells in the lymphoid tissues. The lower quantity of dendritic plasmacytoid cells in those tissues might contribute to the decrease in CD8+ T cells activation. Overall, CXCR3 molecule seems to be an important molecule to be explored during vaccine against Chagas disease strategies.

Chagas disease is caused by Trypanosoma cruzi, a protozoan parasite that has a heterogeneous population composed of a pool of strains with distinct characteristics, including variable levels of virulence. In previous work, transcriptome analyses of parasite genes after infection of human foreskin fibroblasts (HFF) with virulent (CL Brener) and non-virulent (CL-14) clones derived from the CL strain, revealed a reduced expression of genes encoding parasite surface proteins in CL-14 compared to CL Brener during the final steps of the intracellular differentiation from amastigotes to trypomastigotes. Here we analyzed changes in the expression of host genes during in vitro infection of HFF cells with the CL Brener and CL-14 strains by analyzing total RNA extracted from cells at 60 and 96 hours post-infection (hpi) with each strain, as well as from uninfected cells. Similar transcriptome profiles were observed at 60 hpi with both strains compared to uninfected samples. However, at 96 hpi, significant differences in the number and expression levels of several genes, particularly those involved with immune response and cytoskeleton organization, were observed. Further analyses confirmed the difference in the chemokine/cytokine signaling involved with the recruitment and activation of immune cells such as neutrophils upon T. cruzi infection. These findings suggest that infection with the virulent CL Brener strain induces a more robust inflammatory response when compared with the non-virulent CL-14 strain. Importantly, the RNA-Seq data also exposed an unexplored role of fibroblasts as sentinel cells that may act by recruiting neutrophils to the initial site of infection. This role for fibroblasts in the regulation of the inflammatory response during infection by T. cruzi was corroborated by measurements of levels of different chemokines/cytokines during in vitro infection and in plasma from Chagas disease patients as well as by neutrophil activation and migration assays., Author summary Trypanosoma cruzi is the causative agent of Chagas disease, a debilitating and often life-threatening illness that affects 6 to 7 million people mainly in Latin America. The parasite, transmitted to humans by an insect vector, needs to invade different cells from the infected person in order to multiply and spread the infection to various organs, including the heart and the gut. In this study, we investigated how the host cell responds to the infection by analyzing changes in the expression of human genes in fibroblasts infected with the CL Brener and CL-14 strains, which are strains that present highly distinct virulent phenotypes during infection in mice. We showed that human fibroblasts build a strong immune response upon infection by T. cruzi and that this response is different depending on the parasite strain: infection with the virulent CL Brener strain induces a more robust inflammatory response compared with the infection with the avirulent CL-14 strain. We also showed that, in response to the infection, fibroblasts produce molecules that can recruit and activate neutrophils, which are important immune cells that controls the infection.

The leading animal model of experimental Chagas disease, the mouse, plays a significant role in studies for vaccine development, diagnosis, and human therapies. Humans, along with Old World primates, alone among mammals, cannot make the terminal carbohydrate linkage of the α-Gal trisaccharide. It has been established that the anti-α-Gal immune response is likely to be a critical factor for protection against Trypanosoma cruzi (T. cruzi) infection in humans. However, the mice customarily employed for the study of T. cruzi infection naturally express the α-Gal epitope and therefore do not produce anti-α-Gal antibodies. Here, we used the C57BL/6 α-1,3-galactosyltransferase knockout (α-GalT-KO) mouse, which does not express the α-Gal epitope as a model for experimental Chagas disease. We found the anti-α-Gal IgG antibody response to an increase in α-GalT-KO mice infected with Arequipa and Colombiana strains of T. cruzi, leading to fewer parasite nests, lower parasitemia, and an increase of INF-γ, TNF-α, and IL-12 cytokines in the heart of α-GalT-KO mice compared with α-GalT-WT mice on days 60 and 120 postinfection. We therefore agree that the C57BL/6 α-GalT-KO mouse represents a useful model for initial testing of therapeutic and immunological approaches against different strains of T. cruzi.

P. vivax-infected Retics (iRetics) express human leukocyte antigen class I (HLA-I), are recognized by CD8+ T cells and killed by granulysin (GNLY) and granzymes. However, how Plasmodium infection induces MHC-I expression on Retics is unknown. In addition, whether GNLY helps control Plasmodium infection in vivo has not been studied. Here, we examine these questions using rodent infection with the P. yoelii 17XNL strain, which has tropism for Retics. Infection with P. yoelii caused extramedullary erythropoiesis, reticulocytosis and expansion of CD8+CD44+CD62L- IFN-γ-producing T cells that form immune synapses with iRetics. We now provide evidence that MHC-I expression by iRetic is dependent on IFN-γ-induced transcription of IRF-1, MHC-I and β2-microglobulin (β2-m) in erythroblasts. Consistently, CTLs from infected wild type (WT) mice formed immune synapses with iRetics in an IFN-γ- and MHC-I-dependent manner. When challenged with P. yoelii 17XNL, WT mice cleared parasitemia and survived, while IFN-γ KO mice remained parasitemic and all died. β2-m KO mice that do not express MHC-I and have virtually no CD8+ T cells had prolonged parasitemia, and 80% survived. Because mice do not express GNLY, GNLY-transgenic mice can be used to assess the in vivo importance of GNLY. Parasite clearance was accelerated in GNLY-transgenic mice and depletion of CD8+ T cells ablated the GNLY-mediated resistance to P. yoelii. Altogether, our results indicate that in addition to previously described mechanisms, IFN-γ promotes host resistance to the Retic-tropic P. yoelii 17XNL strain by promoting MHC-I expression on iRetics that become targets for CD8+ cytotoxic T lymphocytes and GNLY., Author summary CD8+ cytotoxic T lymphocytes (CTLs) are important for immune defense against intracellular pathogens, such as viruses, bacteria and parasites, and tumor surveillance. CTLs, which recognize peptide epitopes presented by MHC-I molecules expressed in nucleated cells, become activated and kill infected target cells by releasing the contents of cytotoxic granules into the immunological synapse. Since most Plasmodium spp. infect erythrocytes that are enucleated and do not express MHC-I, the role of CD8+ T cells in the blood-stage of malaria has been neglected. We recently showed that P. vivax-infected reticulocytes express MHC-I and are killed in a manner dependent on granulysin (GNLY), a cytotoxic granule effector protein. However, the protective role of CD8+ T cells is controversial and the role of GNLY in vivo remains to be demonstrated. Here, we show that CTLs and GNLY mediate mouse resistance to blood-stage infection with P. yoelii, a rodent malaria parasite that preferably infects reticulocytes.