Search

Summary: Despite successful vaccines and updates, constant mutations of SARS-CoV-2 makes necessary the search for new vaccines. We generated a chimeric protein that comprises the receptor-binding domain from spike and the nucleocapsid antigens (SpiN) from SARS-CoV-2. Once SpiN elicits a protective immune response in rodents, here we show that convalescent and previously vaccinated individuals respond to SpiN. CD4+ and CD8+ T cells from these individuals produced greater amounts of IFN−γ when stimulated with SpiN, compared to SARS-CoV-2 antigens. Also, B cells from these individuals were able to secrete antibodies that recognize SpiN. When administered as a boost dose in mice previously immunized with CoronaVac, ChAdOx1-S or BNT162b2, SpiN was able to induce a greater or equivalent immune response to homologous prime/boost. Our data reveal the ability of SpiN to induce cellular and humoral responses in vaccinated human donors, rendering it a promising candidate.

Abstract Kupffer cells (KCs) are self-maintained tissue-resident macrophages that line liver sinusoids and play an important role on host defense. It has been demonstrated that upon infection or intense liver inflammation, KCs might be severely depleted and replaced by immature monocytic cells; however, the mechanisms of cell death and the alterations on liver immunity against infections deserves further investigation. We explored the impact of acute Plasmodium infection on KC biology and on the hepatic immune response against secondary infections. Similar to patients, infection with Plasmodium chabaudi induced acute liver damage as determined by serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) elevation. This was associated with accumulation of hemozoin, increased of proinflammatory response and impaired bacterial and viral clearance, which led to pathogen spread to other organs. In line with this, mice infected with Plasmodium had enhanced mortality during secondary infections, which was associated with increased production of mitochondrial superoxide, lipid peroxidation and increased free iron within KCs—hallmarks of cell death by ferroptosis. Therefore, we revealed that accumulation of iron with KCs, triggered by uptake of circulating hemozoin, is a novel mechanism of macrophage depletion and liver inflammation during malaria, providing novel insights on host susceptibility to secondary infections. Malaria can cause severe liver damage, along with depletion of liver macrophages, which can predispose individuals to secondary infections and enhance the chances of death.

BackgroundPlasmodium spp. infection triggers the production of inflammatory cytokines that are essential for parasite control, and conversely responsible for symptoms of malaria. Monocytes play a role in host defense against Plasmodium vivax infection and represent the main source of inflammatory cytokines and reactive oxygen species. The anti-inflammatory cytokine IL-10 is a key regulator preventing exacerbated inflammatory responses. Studies suggested that different clinical presentations of malaria are strongly associated with an imbalance in the production of inflammatory and anti-inflammatory cytokines.MethodsA convenience sampling of peripheral blood mononuclear cells from Plasmodium vivax-infected patients and healthy donors were tested for the characterization of cytokine and adenosine production and the expression of ectonucleotidases and purinergic receptors.ResultsHere we show that despite a strong inflammatory response, monocytes also bear a modulatory role during malaria. High levels of IL-10 are produced during P. vivax infection and its production can be triggered in monocytes by P. vivax-infected reticulocytes. Monocytes express high levels of ectonucleotidases, indicating their important role in extracellular ATP modulation and consequently in adenosine production. Plasmatic levels of adenosine are not altered in patients experiencing acute malaria; however, their monocyte subsets displayed an increased expression of P1 purinergic receptors. In addition, adenosine decreases Tumor Necrosis Factor production by monocytes, which was partially abolished with the blockage of the A2a receptor.ConclusionMonocytes have a dual role, attempting to control both the P. vivax infection and the inflammatory response. Purinergic receptor modulators emerge as an untapped approach to ameliorate clinical malaria.

SARS-CoV-2 diagnostic tests have become an important tool for pandemic control. Among the alternatives for COVID-19 diagnosis, antigen rapid diagnostic tests (Ag-RDT) are very convenient and widely used. However, as SARS-CoV-2 variants may continuously emerge, the replacement of tests and reagents may be required to maintain the sensitivity of Ag-RDTs. Here, we describe the development and validation of an Ag-RDT during an outbreak of the Omicron variant, including the characterization of a new monoclonal antibody (anti-DTC-N 1B3 mAb) that recognizes the Nucleocapsid protein (N). The anti-DTC-N 1B3 mAb recognized the sequence TFPPTEPKKDKKK located at the C-terminus of the N protein of main SARS-CoV-2 variants of concern. Accordingly, the Ag-RDT prototypes using the anti-DTC-N 1B3 mAB detected all the SARS-CoV-2 variants—Wuhan, Alpha, Gamma, Delta, P2 and Omicron. The performance of the best prototype (sensitivity of 95.2% for samples with Ct ≤ 25; specificity of 98.3% and overall accuracy of 85.0%) met the WHO recommendations. Moreover, results from a patients’ follow-up study indicated that, if performed within the first three days after onset of symptoms, the Ag-RDT displayed 100% sensitivity. Thus, the new mAb and the Ag-RDT developed herein may constitute alternative tools for COVID-19 point-of-care diagnosis and epidemiological surveillance.

This review does not intend to convey detailed experimental or bibliographic data. Instead, it expresses the informal authors’ personal views on topics that range from basic research on antigens and experimental models for Trypanosoma cruzi infection to vaccine prospects and vaccine production. The review also includes general aspects of Chagas’ disease control and international and national policies on the subject. The authors contributed equally to the paper.

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.

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.

ABSTRACT 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.

[This corrects the article DOI: 10.1371/journal.pntd.0008414.].

Summary: Individuals with malaria exhibit increased morbidity and mortality when infected with Gram-negative (Gr−) bacteria. To explore this experimentally, we performed co-infection of mice with Plasmodium chabaudi and Citrobacter rodentium, an extracellular Gr− bacterial pathogen that infects the large intestine. While single infections are controlled effectively, co-infection results in enhanced virulence that is characterized by prolonged systemic bacterial persistence and high mortality. Mortality in co-infected mice is associated with disrupted iron metabolism, elevated levels of plasma heme, and increased mitochondrial reactive oxygen species (ROS) production by phagocytes. In addition, iron acquisition by the bacterium plays a key role in pathogenesis because co-infection with a mutant C. rodentium strain lacking a critical iron acquisition pathway does not cause mortality. These results indicate that disrupted iron metabolism may drive mortality during co-infection with C. rodentium and P. chabaudi by both altering host immune responses and facilitating bacterial persistence.

The Apicomplexa protozoan Toxoplasma gondii is 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. gondii infection 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 II T. gondii strain, 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) chronic T. gondii infection, 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 chronic T. gondii infection.

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.

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.

The emergence of new SARS-CoV-2 variants represents a constant threat to world public health. The SARS-CoV-2 Delta variant was identified in late 2020 in India; since then, it has spread to many other countries, replacing other predominant lineages and raising concerns about vaccination efficiency. We evaluated the sensitivity of the Delta variant to antibodies elicited by COVID-19 vaccinated (CoronaVac and ChAdOx1) and convalescent individuals previously infected by earlier lineages and by the Gamma variant. No reduction in the neutralizing efficacy of the Delta variant was observed when compared to B lineage and a reduced neutralization was observed for the Gamma variant. Our results indicate that neutralization of the Delta variant is not compromised in individuals vaccinated by CoronaVac or ChAdOx1; however, a reduction in neutralization efficacy is expected for individuals infected by the Gamma variant, highlighting the importance of continuous vaccination even for previously infected individuals.

The persistent circulation of SARS-CoV-2 represents an ongoing global threat due to the emergence of new viral variants that can sometimes evade the immune system of previously exposed or vaccinated individuals. We conducted a follow-up study of adult individuals that had received an inactivated SARS-CoV-2 vaccine, evaluating antibody production and neutralizing activity over a period of 6 months. In addition, we performed mice immunization with inactivated SARS-CoV-2, and evaluated the immune response and pathological outcomes against Gamma and Zeta variant infection. Vaccinated individuals produced high levels of antibodies with robust neutralizing activity, which was significantly reduced against Gamma and Zeta variants. Production of IgG anti-S antibodies and neutralizing activity robustly reduced after 6 months of vaccination. Immunized mice demonstrated cellular response against Gamma and Zeta variants, and after viral infection, reduced viral loads, IL-6 expression, and histopathological outcome in the lungs. TNF levels were unchanged in immunized or not immunized mice after infection with the Gamma variant. Furthermore, serum neutralization activity rapidly increases after infection with the Gamma and Zeta variants. Our data suggest that immunization with inactivated WT SARS-CoV-2 induces a promptly responsive cross-reactive immunity response against the Gamma and Zeta variants, reducing COVID-19 pathological outcomes.

CD8+ T lymphocytes play an important role in controlling infections by intracellular pathogens. Chemokines and their receptors are crucial for the migration of CD8+ T-lymphocytes, which are the main IFNγ producers and cytotoxic effectors cells. Although the participation of chemokine ligands and receptors has been largely explored in viral infection, much less is known in infection by Trypanosoma cruzi, the causative agent of Chagas disease. After T. cruzi infection, CXCR3 chemokine receptor is highly expressed on the surface of CD8+ T-lymphocytes. Here, we hypothesized that CXCR3 is a key molecule for migration of parasite-specific CD8+ T-cells towards infected tissues, where they may play their effector activities. Using a model of induction of resistance to highly susceptible A/Sn mice using an ASP2-carrying DNA/adenovirus prime-boost strategy, we showed that CXCR3 expression was upregulated on CD8+ T-cells, which selectively migrated towards its ligands CXCL9 and CXCL10. Anti-CXCR3 administration reversed the vaccine-induced resistance to T. cruzi infection in a way associated with hampered cytotoxic activity and increased proapoptotic markers on the H2KK-restricted TEWETGQI-specific CD8+ T-cells. Furthermore, CXCR3 receptor critically guided TEWETGQI-specific effector CD8+ T-cells to the infected heart tissue that express CXCL9 and CXCL10. Overall, our study pointed CXCR3 and its ligands as key molecules to drive T. cruzi-specific effector CD8+ T-cells into the infected heart tissue. The unveiling of the process driving cell migration and colonization of infected tissues by pathogen-specific effector T-cells is a crucial requirement to the development of vaccine strategies.

BACKGROUND Visceral Leishmaniasis (VL) is an infectious disease that is a significant cause of death among infants aged under 1 year and the elderly in Brazil. Serodiagnosis is a mainstay of VL elimination programs; however, it has significant limitations due to low accuracy. OBJECTIVE This study aimed to evaluate three recombinant Leishmania infantum proteins (rFc, rC9, and rA2) selected from previous proteomics and genomics analyses to develop enzyme-linked immunosorbent assay (ELISA) and immunochromatographic tests (ICT) for the serodiagnosis of human VL (HVL) and canine VL (CVL). METHODS A total of 186 human (70 L. infantum-infected symptomatic, 20 other disease-infected, and 96 healthy) and 185 canine (82 L. infantum-infected symptomatic, 27 L. infantum-infected asymptomatic, and 76 healthy) sera samples were used for antibody detection. FINDINGS Of the three proteins, rA2 (91.5% sensitivity and 87% specificity) and rC9 (95.7% sensitivity and 87.5% specificity) displayed the best performance in ELISA-HVL and ELISA-CVL, respectively. ICT-rA2 also displayed the best performance for HVL diagnosis (92.3% sensitivity and 88.0% specificity) and had high concordance with immunofluorescence antibody tests (IFAT), ELISA-rK39, IT-LEISH®, and ELISAEXT. ICT-rFc, ICT-rC9, and ICT-rA2 had sensitivities of 88.6%, 86.5%, and 87.0%, respectively, with specificity values of 84.0%, 92.0%, and 100%, respectively for CVL diagnosis. MAIN CONCLUSIONS The three antigens selected by us are promising candidates for VL diagnosis regardless of the test format, although the antigen combinations and test parameters may warrant further optimisation.

Visceral leishmaniasis (VL) is a major public health issue reported as the second illness in mortality among all tropical diseases. Clinical trials have shown that protection against VL is associated with robust T cell responses, especially those producing IFN-γ. The Leishmania amastigote 2 (A2) protein has been repeatedly described as immunogenic and protective against VL in different animal models; it is recognized by human T cells, and it is also commercially available in a vaccine formulation containing saponin against canine VL. Moving toward a more appropriate formulation for human vaccination, here, we tested a new optimized version of the recombinant protein (rA2), designed for Escherichia coli expression, in combination with adjuvants that have been approved for human use. Moreover, aiming at improving the cellular immune response triggered by rA2, we generated a recombinant live vaccine vector using Trypanosoma cruzi CL-14 non-virulent strain, named CL-14 A2. Mice immunized with respective rA2, adsorbed in Alum/CpG B297, a TLR9 agonist recognized by mice and human homologs, or with the recombinant CL-14 A2 parasites through homologous prime-boost protocol, were evaluated for antigen-specific immune responses and protection against Leishmania infantum promastigote challenge. Immunization with the new rA2/Alum/CpG formulations and CL-14 A2 transgenic vectors elicited stronger cellular immune responses than control groups, as shown by increased levels of IFN-γ, conferring protection against L. infantum challenge. Interestingly, the use of the wild-type CL-14 alone was enough to boost immunity and confer protection, confirming the previously reported immunogenic potential of this strain. Together, these results support the success of both the newly designed rA2 antigen and the ability of T. cruzi CL-14 to induce strong T cell-mediated immune responses against VL in animal models when used as a live vaccine vector. In conclusion, the vaccination strategies explored here reveal promising alternatives for the development of new rA2 vaccine formulations to be translated human clinical trials.

Neutrophils are the most abundant leukocyte population in the bloodstream, the primary compartment of Plasmodium sp. infection. However, the role of these polymorphonuclear cells in mediating either the resistance or the pathogenesis of malaria is poorly understood. We report that circulating neutrophils from malaria patients are highly activated, as indicated by a strong type I interferon transcriptional signature, increased expression of surface activation markers, enhanced release of reactive oxygen species and myeloperoxidase, and a high frequency of low-density granulocytes. The activation of neutrophils was associated with increased levels of serum alanine and aspartate aminotransferases, indicating liver damage. In a rodent malaria model, we observed intense recruitment of neutrophils to liver sinusoids. Neutrophil migration and IL-1β and chemokine expression as well as liver damage were all dependent on type I interferon signaling. The data suggest that type I interferon signaling has a central role in neutrophil activation and malaria pathogenesis.

Integrins mediate the lymphocyte migration into an infected tissue, and these cells are essential for controlling the multiplication of many intracellular parasites such as Trypanosoma cruzi, the causative agent of Chagas disease. Here, we explore LFA-1 and VLA-4 roles in the migration of specific CD8+ T cells generated by heterologous prime-boost immunization during experimental infection with T. cruzi. To this end, vaccinated mice were treated with monoclonal anti-LFA-1 and/or anti-VLA-4 to block these molecules. After anti-LFA-1, but not anti-VLA-4 treatment, all vaccinated mice displayed increased blood and tissue parasitemia, and quickly succumbed to infection. In addition, there was an accumulation of specific CD8+ T cells in the spleen and lymph nodes and a decrease in the number of those cells, especially in the heart, suggesting that LFA-1 is important for the output of specific CD8+ T cells from secondary lymphoid organs into infected organs such as the heart. The treatment did not alter CD8+ T cell effector functions such as the production of pro-inflammatory cytokines and granzyme B, and maintained the proliferative capacity after treatment. However, the specific CD8+ T cell direct cytotoxicity was impaired after LFA-1 blockade. Also, these cells expressed higher levels of Fas/CD95 on the surface, suggesting that they are susceptible to programmed cell death by the extrinsic pathway. We conclude that LFA-1 plays an important role in the migration of specific CD8+ T cells and in the direct cytotoxicity of these cells.

Although the importance of humoral immunity to malaria has been established, factors that control antibody production are poorly understood. Follicular helper T cells (Tfh cells) are pivotal for generating high-affinity, long-lived antibody responses. While it has been proposed that expansion of antigen-specific Tfh cells, interleukin (IL) 21 production and robust germinal center formation are associated with protection against malaria in mice, whether Tfh cells are found during Plasmodium vivax (P. vivax) infection and if they play a role during disease remains unknown. Our goal was to define the role of Tfh cells during P. vivax malaria. We demonstrate that P. vivax infection triggers IL-21 production and an increase in Tfh cells (PD-1+ICOS+CXCR5+CD45RO+CD4+CD3+). As expected, FACS-sorted Tfh cells, the primary source of IL-21, induced immunoglobulin production by purified naïve B cells. Furthermore, we found that P. vivax infection alters the B cell compartment and these alterations were dependent on the number of previous infections. First exposure leads to increased proportions of activated and atypical memory B cells and decreased frequencies of classical memory B cells, whereas patients that experienced multiple episodes displayed lower proportions of atypical B cells and higher frequencies of classical memory B cells. Despite the limited sample size, but consistent with the latter finding, the data suggest that patients who had more than five infections harbored more Tfh cells and produce more specific antibodies. P. vivax infection triggers IL-21 production by Tfh that impact B cell responses in humans.

To assess reinfection of BALB/c mice with different Toxoplasma gondii strains, the animals were prime infected with the non-virulent D8 strain and challenged with virulent recombinant strains. Thirty days after challenge, brain cysts were obtained from surviving BALB/c mice and inoculated in Swiss mice to obtain tachyzoites for DNA extraction and PCR-RFLP analysis to distinguish the different T. gondii strains present in possible co-infections. Anti-Toxoplasma immune responses were evaluated in D8-primed BALB/c mice by detecting IFN-³ and IL-10 produced by T cells and measuring immunoglobulin levels in serum samples. PCR-RFLP demonstrated that BALB/c mice were reinfected with the EGS strain at 45 days post prime infection (dpi) and with the EGS and CH3 strains at 180 dpi. High levels of IFN-³ were detected after D8 infection, with no significant difference between 45 and 180-day intervals. However, higher IL-10 levels and higher plasmatic IgG1 and IgA were detected from samples obtained 180 days after infection. BALB/c mice were susceptible to reinfection with different recombinant T. gondii strains and this susceptibility correlated with enhancement of IL-10 production.

In Chagas disease, understanding how the immune response controls parasite growth but also leads to heart damage may provide insight into the design of new therapeutic strategies. Tumor necrosis factor-alpha (TNF-α) is important for resistance to acute Trypanosoma cruzi infection; however, in patients suffering from chronic T. cruzi infection, plasma TNF-α levels correlate with cardiomyopathy. Recent data suggest that CD8-enriched chagasic myocarditis formation involves CCR1/CCR5-mediated cell migration. Herein, the contribution of TNF-α, especially signaling through the receptor TNFR1/p55, to the pathophysiology of T. cruzi infection was evaluated with a focus on the development of myocarditis and heart dysfunction. Colombian strain-infected C57BL/6 mice had increased frequencies of TNFR1/p55+ and TNF-α+ splenocytes. Although TNFR1-/- mice exhibited reduced myocarditis in the absence of parasite burden, they succumbed to acute infection. Similar to C57BL/6 mice, Benznidazole-treated TNFR1-/- mice survived acute infection. In TNFR1-/- mice, reduced CD8-enriched myocarditis was associated with defective activation of CD44+CD62Llow/- and CCR5+ CD8+ lymphocytes. Also, anti-TNF-α treatment reduced the frequency of CD8+CCR5+ circulating cells and myocarditis, though parasite load was unaltered in infected C3H/HeJ mice. TNFR1-/- and anti-TNF-α-treated infected mice showed regular expression of connexin-43 and reduced fibronectin deposition, respectively. Furthermore, anti-TNF-α treatment resulted in lower levels of CK-MB, a cardiomyocyte lesion marker. Our results suggest that TNF/TNFR1 signaling promotes CD8-enriched myocarditis formation and heart tissue damage, implicating the TNF/TNFR1 signaling pathway as a potential therapeutic target for control of T. cruzi-elicited cardiomyopathy.

The inflammatory cytokine interferon-gamma (IFNγ) is crucial for immunity against intracellular pathogens such as the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (CD). IFNγ is a pleiotropic cytokine which regulates activation of immune and non-immune cells; however, the effect of IFNγ in the central nervous system (CNS) and astrocytes during CD is unknown. Here we show that parasite persists in the CNS of C3H/He mice chronically infected with the Colombian T. cruzi strain despite the increased expression of IFNγ mRNA. Furthermore, most of the T. cruzi-bearing cells were astrocytes located near IFNγ+ cells. Surprisingly, in vitro experiments revealed that pretreatment with IFNγ promoted the infection of astrocytes by T. cruzi increasing uptake and proliferation of intracellular forms, despite inducing increased production of nitric oxide (NO). Importantly, the effect of IFNγ on T. cruzi uptake and growth is completely blocked by the anti-tumor necrosis factor (TNF) antibody Infliximab and partially blocked by the inhibitor of nitric oxide synthesis L-NAME. These data support that IFNγ fuels astrocyte infection by T. cruzi and critically implicate IFNγ-stimulated T. cruzi-infected astrocytes as sources of TNF and NO, which may contribute to parasite persistence and CNS pathology in CD.

Chagas disease (CD), caused by the protozoan Trypanosoma cruzi, is a prototypical neglected tropical disease. Specific immunity promotes acute phase survival. Nevertheless, one-third of CD patients develop chronic chagasic cardiomyopathy (CCC) associated with parasite persistence and immunological unbalance. Currently, the therapeutic management of patients only mitigates CCC symptoms. Therefore, a vaccine arises as an alternative to stimulate protective immunity and thereby prevent, delay progression and even reverse CCC. We examined this hypothesis by vaccinating mice with replication-defective human Type 5 recombinant adenoviruses (rAd) carrying sequences of amastigote surface protein-2 (rAdASP2) and trans-sialidase (rAdTS) T. cruzi antigens. For prophylactic vaccination, naïve C57BL/6 mice were immunized with rAdASP2+rAdTS (rAdVax) using a homologous prime/boost protocol before challenge with the Colombian strain. For therapeutic vaccination, rAdVax administration was initiated at 120 days post-infection (dpi), when mice were afflicted by CCC. Mice were analyzed for electrical abnormalities, immune response and cardiac parasitism and tissue damage. Prophylactic immunization with rAdVax induced antibodies and H-2Kb-restricted cytotoxic and interferon (IFN)γ-producing CD8+ T-cells, reduced acute heart parasitism and electrical abnormalities in the chronic phase. Therapeutic vaccination increased survival and reduced electrical abnormalities after the prime (analysis at 160 dpi) and the boost (analysis at 180 and 230 dpi). Post-therapy mice exhibited less heart injury and electrical abnormalities compared with pre-therapy mice. rAdVax therapeutic vaccination preserved specific IFNγ-mediated immunity but reduced the response to polyclonal stimuli (anti-CD3 plus anti-CD28), CD107a+ CD8+ T-cell frequency and plasma nitric oxide (NO) levels. Moreover, therapeutic rAdVax reshaped immunity in the heart tissue as reduced the number of perforin+ cells, preserved the number of IFNγ+ cells, increased the expression of IFNγ mRNA but reduced inducible NO synthase mRNA. Vaccine-based immunostimulation with rAd might offer a rational alternative for re-programming the immune response to preserve and, moreover, recover tissue injury in Chagas' heart disease.

Chagas disease, which is caused by the parasite Trypanosoma cruzi, is an important cause of heart failure. We investigated modifications in the cellular electrophysiological and calcium-handling characteristics of an infected mouse heart during the chronic phase of the disease. The patch-clamp technique was used to record action potentials (APs) and L-type Ca2+ and transient outward K+ currents. [Ca2+]i changes were determined using confocal microscopy. Infected ventricular cells showed prolonged APs, reduced transient outward K+ and L-type Ca2+ currents and reduced Ca2+ release from the sarcoplasmic reticulum. Thus, the chronic phase of Chagas disease is characterised by cardiomyocyte dysfunction, which could lead to heart failure.

Chagas disease, which is caused by the parasite Trypanosoma cruzi, is an important cause of heart failure. We investigated modifications in the cellular electrophysiological and calcium-handling characteristics of an infected mouse heart during the chronic phase of the disease. The patch-clamp technique was used to record action potentials (APs) and L-type Ca2+ and transient outward K+ currents. [Ca2+]i changes were determined using confocal microscopy. Infected ventricular cells showed prolonged APs, reduced transient outward K+ and L-type Ca2+ currents and reduced Ca2+ release from the sarcoplasmic reticulum. Thus, the chronic phase of Chagas disease is characterised by cardiomyocyte dysfunction, which could lead to heart failure.

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi (T. cruzi), is characterized by immunopathology driven by IFN-γ secreting Th1-like T cells. T. cruzi has a thick coat of mucin-like glycoproteins covering its surface, which plays an important role in parasite invasion and host immunomodulation. It has been extensively described that T. cruzi or its products—like GPI anchors isolated from GPI-anchored mucins from the trypomastigote life cycle stage (tGPI-mucins)—are potent inducers of proinflammatory responses (i.e., cytokines and NO production) by IFN-γ primed murine macrophages. However, little is known about whether T. cruzi or GPI-mucins exert a similar action in human cells. We therefore decided to further investigate the in vitro cytokine production profile from human mononuclear cells from uninfected donors exposed to T. cruzi as well as tGPI-mucins. We observed that both living T. cruzi trypomastigotes and tGPI-mucins are potent inducers of IL-12 by human peripheral blood monocytes and this effect depends on CD40-CD40L interaction and IFN-γ. Our findings suggest that the polarized T1-type cytokine profile seen in T. cruzi infected patients might be a long-term effect of IL-12 production induced by lifelong exposure to T. cruzi tGPI-mucins.

Recombinant influenza viruses are promising viral platforms to be used as antigen delivery vectors. To this aim, one of the most promising approaches consists of generating recombinant viruses harboring partially truncated neuraminidase (NA) segments. To date, all studies have pointed to safety and usefulness of this viral platform. However, some aspects of the inflammatory and immune responses triggered by those recombinant viruses and their safety to immunocompromised hosts remained to be elucidated. In the present study, we generated a recombinant influenza virus harboring a truncated NA segment (vNA-Δ) and evaluated the innate and inflammatory responses and the safety of this recombinant virus in wild type or knock-out (KO) mice with impaired innate (Myd88 -/-) or acquired (RAG -/-) immune responses. Infection using truncated neuraminidase influenza virus was harmless regarding lung and systemic inflammatory response in wild type mice and was highly attenuated in KO mice. We also demonstrated that vNA-Δ infection does not induce unbalanced cytokine production that strongly contributes to lung damage in infected mice. In addition, the recombinant influenza virus was able to trigger both local and systemic virus-specific humoral and CD8+ T cellular immune responses which protected immunized mice against the challenge with a lethal dose of homologous A/PR8/34 influenza virus. Taken together, our findings suggest and reinforce the safety of using NA deleted influenza viruses as antigen delivery vectors against human or veterinary pathogens.

In the present study we evaluated the protection raised by immunization with recombinant influenza viruses carrying sequences coding for polypeptides corresponding to medial and carboxi-terminal moieties of Trypanosoma cruzi ´s amastigote surface protein 2 (ASP2). Those viruses were used in sequential immunization with recombinant adenovirus (heterologous prime-boost immunization protocol) encoding the complete sequence of ASP2 (Ad-ASP2) in two mouse strains (C57BL/6 and C3H/He). The CD8 effector response elicited by this protocol was comparable to that observed in mice immunized twice with Ad-ASP2 and more robust than that observed in mice that were immunized once with Ad-ASP2. Whereas a single immunization with Ad-ASP2 sufficed to completely protect C57BL/6 mice, a higher survival rate was observed in C3H/He mice that were primed with recombinant influenza virus and boosted with Ad-ASP2 after being challenged with T. cruzi. Analyzing the phenotype of CD8+ T cells obtained from spleen of vaccinated C3H/He mice we observed that heterologous prime-boost immunization protocol elicited more CD8+ T cells specific for the immunodominant epitope as well as a higher number of CD8+ T cells producing TNF-α and IFN-γ and a higher mobilization of surface marker CD107a. Taken together, our results suggest that immunodominant subpopulations of CD8+ T elicited after immunization could be directly related to degree of protection achieved by different immunization protocols using different viral vectors. Overall, these results demonstrated the usefulness of recombinant influenza viruses in immunization protocols against Chagas Disease.

The use of recombinant viral vectors expressing T. gondii antigens is a safe and efficient approach to induce immune response against the parasite and a valuable tool for vaccine development. We have previously protected mice from toxoplasmosis by immunizing the animals with an adenovirus expressing the protein SAG1 (AdSAG1) of T. gondii. We are now looking for ways to improve the vaccination strategy and enhance protection. One limitation of homologous vaccinations (sequential doses of the same vector) is induction of anti-vector immune response that blocks cell transduction, restricts transgene expression and, consequently, compromises the overall outcome of vaccination. One way to avert the effects of anti-vector response is to use different viruses in prime and boost (heterologous vaccination). Bearing this in mind, we generated a modified Vaccinia Virus Ankara encoding SAG1 (MVASAG1), to be tested as boost agent after prime with AdSAG1. Although minor differences were observed in the magnitude of the anti-SAG1 immune response induced by each vaccination protocol, the heterologous immunization with AdSAG1 followed by MVASAG1 resulted in improved capacity to control brain cyst formation in a model of chronic toxoplasmosis in C57BL/6 mice.

BACKGROUND: The factors influencing variation in the clinical forms of Chagas disease have not been elucidated; however, it is likely that the genetics of both the host and the parasite are involved. Several studies have attempted to correlate the T. cruzi strains involved in infection with the clinical forms of the disease by using hemoculture and/or PCR-based genotyping of parasites from infected human tissues. However, both techniques have limitations that hamper the analysis of large numbers of samples. The goal of this work was to identify conserved and polymorphic linear B-cell epitopes of T. cruzi that could be used for serodiagnosis and serotyping of Chagas disease using ELISA. METHODOLOGY: By performing B-cell epitope prediction on proteins derived from pair of alleles of the hybrid CL Brener genome, we have identified conserved and polymorphic epitopes in the two CL Brener haplotypes. The rationale underlying this strategy is that, because CL Brener is a recent hybrid between the TcII and TcIII DTUs (discrete typing units), it is likely that polymorphic epitopes in pairs of alleles could also be polymorphic in the parental genotypes. We excluded sequences that are also present in the Leishmania major, L. infantum, L. braziliensis and T. brucei genomes to minimize the chance of cross-reactivity. A peptide array containing 150 peptides was covalently linked to a cellulose membrane, and the reactivity of the peptides was tested using sera from C57BL/6 mice chronically infected with the Colombiana (TcI) and CL Brener (TcVI) clones and Y (TcII) strain. FINDINGS AND CONCLUSIONS: A total of 36 peptides were considered reactive, and the cross-reactivity among the strains is in agreement with the evolutionary origin of the different T. cruzi DTUs. Four peptides were tested against a panel of chagasic patients using ELISA. A conserved peptide showed 95.8% sensitivity, 88.5% specificity, and 92.7% accuracy for the identification of T. cruzi in patients infected with different strains of the parasite. Therefore, this peptide, in association with other T. cruzi antigens, may improve Chagas disease serodiagnosis. Together, three polymorphic epitopes were able to discriminate between the three parasite strains used in this study and are thus potential targets for Chagas disease serotyping.

BackgroundTrypanosoma cruzi is the etiological agent of Chagas disease, a debilitating illness that affects millions of people in the Americas. A major finding of the T. cruzi genome project was the discovery of a novel multigene family composed of approximately 1,300 genes that encode mucin-associated surface proteins (MASPs). The high level of polymorphism of the MASP family associated with its localization at the surface of infective forms of the parasite suggests that MASP participates in host-parasite interactions. We speculate that the large repertoire of MASP sequences may contribute to the ability of T. cruzi to infect several host cell types and/or participate in host immune evasion mechanisms.MethodsBy sequencing seven cDNA libraries, we analyzed the MASP expression profile in trypomastigotes derived from distinct host cells and after sequential passages in acutely infected mice. Additionally, to investigate the MASP antigenic profile, we performed B-cell epitope prediction on MASP proteins and designed a MASP-specific peptide array with 110 putative epitopes, which was screened with sera from acutely infected mice.Findings and conclusionsWe observed differential expression of a few MASP genes between trypomastigotes derived from epithelial and myoblast cell lines. The more pronounced MASP expression changes were observed between bloodstream and tissue-culture trypomastigotes and between bloodstream forms from sequential passages in acutely infected mice. Moreover, we demonstrated that different MASP members were expressed during the acute T. cruzi infection and constitute parasite antigens that are recognized by IgG and IgM antibodies. We also found that distinct MASP peptides could trigger different antibody responses and that the antibody level against a given peptide may vary after sequential passages in mice. We speculate that changes in the large repertoire of MASP antigenic peptides during an infection may contribute to the evasion of host immune responses during the acute phase of Chagas disease.

BACKGROUND: Zoonotic visceral leishmaniasis (VL) is a severe infectious disease caused by protozoan parasites of the genus Leishmania and the domestic dogs are the main urban parasite reservoir hosts. In Brazil, indirect fluorescence antibody tests (IFAT) and indirect enzyme linked immunosorbent assay (ELISA) using promastigote extracts are widely used in epidemiological surveys. However, their sensitivity and specificity have often been compromised by the use of complex mixtures of antigens, which reduces their accuracy allowing the maintenance of infected animals that favors transmission to humans. In this context, the use of combinations of defined peptides appears favorable. Therefore, they were tested by combinations of five peptides derived from the previously described Leishmania diagnostic antigens A2, NH, LACK and K39. METHODOLOGY/PRINCIPAL FINDINGS: Combinations of peptides derived A2, NH, LACK and K39 antigens were used in ELISA with sera from 44 human patients and 106 dogs. Improved sensitivities and specificities, close to 100%, were obtained for both sera of patients and dogs. Moreover, high sensitivity and specificity were observed even for canine sera presenting low IFAT anti-Leishmania antibody titers or from asymptomatic animals. CONCLUSIONS/SIGNIFICANCE: The use of combinations of B cell predicted synthetic peptides derived from antigens A2, NH, LACK and K39 may provide an alternative for improved sensitivities and specificities for immunodiagnostic assays of VL.

In Chagas disease, CD8(+) T-cells are critical for the control of Trypanosoma cruzi during acute infection. Conversely, CD8(+) T-cell accumulation in the myocardium during chronic infection may cause tissue injury leading to chronic chagasic cardiomyopathy (CCC). Here we explored the role of CD8(+) T-cells in T. cruzi-elicited heart injury in C57BL/6 mice infected with the Colombian strain. Cardiomyocyte lesion evaluated by creatine kinase-MB isoenzyme activity levels in the serum and electrical abnormalities revealed by electrocardiogram were not associated with the intensity of heart parasitism and myocarditis in the chronic infection. Further, there was no association between heart injury and systemic anti-T. cruzi CD8(+) T-cell capacity to produce interferon-gamma (IFNγ) and to perform specific cytotoxicity. Heart injury, however, paralleled accumulation of anti-T. cruzi cells in the cardiac tissue. In T. cruzi infection, most of the CD8(+) T-cells segregated into IFNγ(+) perforin (Pfn)(neg) or IFNγ(neg)Pfn(+) cell populations. Colonization of the cardiac tissue by anti-T. cruzi CD8(+)Pfn(+) cells paralleled the worsening of CCC. The adoptive cell transfer to T. cruzi-infected cd8(-/-) recipients showed that the CD8(+) cells from infected ifnγ(-/-)pfn(+/+) donors migrate towards the cardiac tissue to a greater extent and caused a more severe cardiomyocyte lesion than CD8(+) cells from ifnγ(+/+)pfn(-/-) donors. Moreover, the reconstitution of naïve cd8(-/-) mice with CD8(+) cells from naïve ifnγ(+/+)pfn(-/-) donors ameliorated T. cruzi-elicited heart injury paralleled IFNγ(+) cells accumulation, whereas reconstitution with CD8(+) cells from naïve ifnγ(-/-)pfn(+/+) donors led to an aggravation of the cardiomyocyte lesion, which was associated with the accumulation of Pfn(+) cells in the cardiac tissue. Our data support a possible antagonist effect of CD8(+)Pfn(+) and CD8(+)IFNγ(+) cells during CCC. CD8(+)IFNγ(+) cells may exert a beneficial role, whereas CD8(+)Pfn(+) may play a detrimental role in T. cruzi-elicited heart injury.

BACKGROUND: The factors contributing to chronic Chagas' heart disease remain unknown. High nitric oxide (NO) levels have been shown to be associated with cardiomyopathy severity in patients. Further, NO produced via inducible nitric oxide synthase (iNOS/NOS2) is proposed to play a role in Trypanosoma cruzi control. However, the participation of iNOS/NOS2 and NO in T. cruzi control and heart injury has been questioned. Here, using chronically infected rhesus monkeys and iNOS/NOS2-deficient (Nos2(-/-)) mice we explored the participation of iNOS/NOS2-derived NO in heart injury in T. cruzi infection. METHODOLOGY: Rhesus monkeys and C57BL/6 and Nos2(-/-) mice were infected with the Colombian T. cruzi strain. Parasite DNA was detected by polymerase chain reaction, T. cruzi antigens and iNOS/NOS2(+) cells were immunohistochemically detected in heart sections and NO levels in serum were determined by Griess reagent. Heart injury was assessed by electrocardiogram (ECG), echocardiogram (ECHO), creatine kinase heart isoenzyme (CK-MB) activity levels in serum and connexin 43 (Cx43) expression in the cardiac tissue. RESULTS: Chronically infected monkeys presented conduction abnormalities, cardiac inflammation and fibrosis, which resembled the spectrum of human chronic chagasic cardiomyopathy (CCC). Importantly, chronic myocarditis was associated with parasite persistence. Moreover, Cx43 loss and increased CK-MB activity levels were primarily correlated with iNOS/NOS2(+) cells infiltrating the cardiac tissue and NO levels in serum. Studies in Nos2(-/-) mice reinforced that the iNOS/NOS2-NO pathway plays a pivotal role in T. cruzi-elicited cardiomyocyte injury and in conduction abnormalities that were associated with Cx43 loss in the cardiac tissue. CONCLUSION: T. cruzi-infected rhesus monkeys reproduce features of CCC. Moreover, our data support that in T. cruzi infection persistent parasite-triggered iNOS/NOS2 in the cardiac tissue and NO overproduction might contribute to CCC severity, mainly disturbing of the molecular pathway involved in electrical synchrony. These findings open a new avenue for therapeutic tools in Chagas' heart disease.