Expression of Inhibitory Receptors TIGIT, TIM-3, and LAG-3 on CD4+ T Cells from Patients with Different Clinical Forms of Chronic Chagas Disease.

T cells are central to the adaptive immune response against Trypanosoma cruzi infection. In chronic Chagas disease (CCD), circulating parasite-specific memory T cells show reduced functionality and increased expression of inhibitory receptors as a result of persistent antigenic stimulation. This phenotype has been linked to progression of cardiac pathology, whereas the presence of polyfunctional T cells shows association with therapeutic success. In this study, we demonstrate that T. cruzi-specific human CD4+ T cells can be identified by their expression of OX40 and CD25 upon in vitro stimulation. We characterized the expression of the inhibitory receptors T cell immunoreceptor with Ig and ITIM domains (TIGIT), T cell Ig and mucin-domain containing-3 (TIM-3), and lymphocyte activation gene 3 (LAG-3) in CD4+ T cells from CCD patients with and without cardiac alterations. Our results show that, independently of their clinical stage, CCD patients present an increased frequency of CD4+ T cells expressing TIGIT in comparison with non-T. cruzi-infected donors. Exposure to parasite Ags increases the expression of TIM-3 in CD4+ T cells from CCD patients, especially in those with cardiac compromise. Upregulation of LAG-3 was also detected in CCD individuals without cardiac manifestations, predominantly within the subpopulation of cells that did not become activated upon stimulation. Further differences were found between groups in the coexpression of these receptors. Blockade of each individual receptor did not affect activation or the production of IFN-γ and IL-10 by CD4+ T cells in response to parasite Ags. Our results suggest a role for TIGIT, TIM-3, and LAG-3 in the modulation of inflammatory phenomena thought to ultimately lead to tissue damage and cardiac pathology.

Characterization of Novel Trypanosoma cruzi-Specific Antigen with Potential Use in the Diagnosis of Chagas Disease.

Chagas disease is caused by the parasite Trypanosoma cruzi. In humans, it evolves into a chronic disease, eventually resulting in cardiac, digestive, and/or neurological disorders. In the present study, we characterized a novel T. cruzi antigen named Tc323 (TcCLB.504087.20), recognized by a single-chain monoclonal antibody (scFv 6B6) isolated from the B cells of patients with cardiomyopathy related to chronic Chagas disease. Tc323, a ~323 kDa protein, is an uncharacterized protein showing putative quinoprotein alcohol dehydrogenase-like domains. A computational molecular docking study revealed that the scFv 6B6 binds to an internal domain of Tc323. Immunofluorescence microscopy and Western Blot showed that Tc323 is expressed in the main developmental forms of T. cruzi, localized intracellularly and exhibiting a membrane-associated pattern. According to phylogenetic analysis, Tc323 is highly conserved throughout evolution in all the lineages of T. cruzi so far identified, but it is absent in Leishmania spp. and Trypanosoma brucei. Most interestingly, only plasma samples from patients infected with T. cruzi and those with mixed infection with Leishmania spp. reacted against Tc323. Collectively, our findings demonstrate that Tc323 is a promising candidate for the differential serodiagnosis of chronic Chagas disease in areas where T. cruzi and Leishmania spp. infections coexist.

Activation-induced marker assays for identification of Trypanosoma cruzi-specific CD4 or CD8 T cells in chronic Chagas disease patients.

Antigen-specific T cells are central to the adaptive immune response against T. cruzi infection and underpin the efficacy of on-going vaccine strategies. In this context, the present study focuses on T-cell assays that define the parasite-specificity on the basis of upregulation of TCR stimulation-induced surface markers. For this purpose, we tested different dual marker combinations (OX40, CD25, CD40L, CD137, CD69, PD-L1, CD11a, CD49d, HLA-DR, CD38) to reliably identify activated CD4+ and CD8+ T-cell populations from PBMCs of chronic Chagas disease (CCD) patients after 12 or 24 h of stimulation with T. cruzi lysate. Results demonstrated that activation-induced markers (AIM) assays combining the expression of OX40, CD25, CD40L, CD137, CD69 and/or PD-L1 surface markers are efficient at detecting T. cruzi-specific CD4+ T cells in CCD patients, in comparison to non-infected donors, after both stimulation times. For CD8+ T cells, only PD-L1/OX40 after 24 h of antigen exposure resulted to be useful to track a parasite-specific response. We also demonstrated that the agnostic activation is mediated by different T. cruzi strains, such as Dm28c, CL Brener or Sylvio. Additionally, we successfully used this approach to identify the phenotype of activated T lymphocytes based on the expression of CD45RA and CCR7. Overall, our results show that different combinations of AIM markers represent an effective and simple tool for the detection of T. cruzi-specific CD4+ and CD8+ T cells.

In Silico Guided Discovery of Novel Class I and II Trypanosoma cruzi Epitopes Recognized by T Cells from Chagas' Disease Patients.

T cell-mediated immune response plays a crucial role in controlling Trypanosoma cruzi infection and parasite burden, but it is also involved in the clinical onset and progression of chronic Chagas' disease. Therefore, the study of T cells is central to the understanding of the immune response against the parasite and its implications for the infected organism. The complexity of the parasite-host interactions hampers the identification and characterization of T cell-activating epitopes. We approached this issue by combining in silico and in vitro methods to interrogate patients' T cells specificity. Fifty T. cruzi peptides predicted to bind a broad range of class I and II HLA molecules were selected for in vitro screening against PBMC samples from a cohort of chronic Chagas' disease patients, using IFN-γ secretion as a readout. Seven of these peptides were shown to activate this type of T cell response, and four out of these contain class I and II epitopes that, to our knowledge, are first described in this study. The remaining three contain sequences that had been previously demonstrated to induce CD8+ T cell response in Chagas' disease patients, or bind HLA-A*02:01, but are, in this study, demonstrated to engage CD4+ T cells. We also assessed the degree of differentiation of activated T cells and looked into the HLA variants that might restrict the recognition of these peptides in the context of human T. cruzi infection.

The cytosolic tryparedoxin peroxidase from Trypanosoma cruzi induces a pro-inflammatory Th1 immune response in a peroxidatic cysteine-dependent manner.

Trypanosoma cruzi cytosolic tryparedoxin peroxidase (c-TXNPx) is a 2-Cys peroxiredoxin (Prx) with an important role in detoxifying host cell oxidative molecules during parasite infection. c-TXNPx is a virulence factor, as its overexpression enhances parasite infectivity and resistance to exogenous oxidation. As Prxs from other organisms possess immunomodulatory properties, we studied the effects of c-TXNPx in the immune response and analysed whether the presence of the peroxidatic cysteine is necessary to mediate these properties. To this end, we used a recombinant c-TXNPx and a mutant version (c-TXNPxC52S) lacking the peroxidatic cysteine. We first analysed the oligomerization profile, oxidation state and peroxidase activity of both proteins by gel filtration, Western blot and enzymatic assay, respectively. To investigate their immunological properties, we analysed the phenotype and functional activity of macrophage and dendritic cells and the T-cell response by flow cytometry after injection into mice. Our results show that c-TXNPx, but not c-TXNPxC52S, induces the recruitment of IL-12/23p40-producing innate antigen-presenting cells and promotes a strong specific Th1 immune response. Finally, we studied the cellular and humoral immune response developed in the context of parasite natural infection and found that only wild-type c-TXNPx induces proliferation and high levels of IFN-γ secretion in PBMC from chronic patients without demonstrable cardiac manifestations. In conclusion, we demonstrate that c-TXNPx possesses pro-inflammatory properties that depend on the presence of peroxidatic cysteine that is essential for peroxidase activity and quaternary structure of the protein and could contribute to rational design of immune-based strategies against Chagas disease.

Evaluation of the immune response against Trypanosoma cruzi cytosolic tryparedoxin peroxidase in human natural infection.

Trypanosoma cruzi, the aetiological agent of Chagas disease, has a highly efficient detoxification system to deal with the oxidative burst imposed by its host. One of the antioxidant enzymes involved is the cytosolic tryparedoxin peroxidase (c-TXNPx), which catalyses the reduction to hydrogen peroxide, small-chain organic hydroperoxides and peroxynitrite. This enzyme is present in all parasite stages, and its overexpression renders parasites more resistant to the oxidative defences of macrophages, favouring parasite survival. This work addressed the study of the specific humoral and cellular immune response triggered by c-TXNPx in human natural infection. Thus, sera and peripheral blood mononuclear cells (PBMC) were collected from chronically infected asymptomatic and cardiac patients, and non-infected individuals. Results showed that levels of IgG antibodies against c-TXNPx were low in sera from individuals across all groups. B-cell epitope prediction limited immunogenicity to a few, small regions on the c-TXNPx sequence. At a cellular level, PBMC from asymptomatic and cardiac patients proliferated and secreted interferon-γ after c-TXNPx stimulation, compared with mock control. However, only proliferation was higher in asymptomatic patients compared with cardiac and non-infected individuals. Furthermore, asymptomatic patients showed an enhanced frequency of CD19+ CD69+ cells upon exposure to c-TXNPx. Overall, our results show that c-TXNPx fails to induce a strong immune response in natural infection, being measurable only in those patients without any clinical symptoms. The low impact of c-TXNPx in the human immune response could be strategic for parasite survival, as it keeps this crucial antioxidant enzyme activity safe from the mechanisms of adaptive immune response.

Serological based monitoring of a cohort of patients with chronic Chagas disease treated with benznidazole in a highly endemic area of northern Argentina.

This study aimed to evaluate well-documented diagnostic antigens, named B13, 1F8 and JL7 recombinant proteins, as potential markers of seroconversion in treated chagasic patients. Prospective study, involving 203 patients treated with benznidazole, was conducted from endemic areas of northern Argentina. Follow-up was possible in 107 out of them and blood samples were taken for serology and PCR assays before and 2, 3, 6, 12, 24 and 36 months after treatment initiation. Reactivity against Trypanosoma cruzi lysate and recombinant antigens was measured by ELISA. The rate of decrease of antibody titers showed nonlinear kinetics with an abrupt drop within the first three months after initiation of treatment for all studied antigens, followed by a plateau displaying a low decay until the end of follow-up. At this point, anti-B13, anti-1F8 and anti-JL7 titers were relatively close to the cut-off line, while anti-T. cruzi antibodies still remained positive. At baseline, 60.8% (45/74) of analysed patients tested positive for parasite DNA by PCR and during the follow-up period in 34 out of 45 positive samples (75.5%) could not be detected T. cruzi DNA. Our results suggest that these antigens might be useful as early markers for monitoring antiparasitic treatment in chronic Chagas disease.

Regulatory volume increase and regulatory volume decrease responses in HL-1 atrial myocytes.

BACKGROUND/AIMS: we have investigated whether cultured cardiomyocytes of the cell line HL-1 have the ability to perform regulatory volume responses both in hypotonic and hypertonic conditions. Furthermore, we characterized those regulatory responses and studied the effects of bumetanide and DIDS in volume regulation of HL-1 cells. METHODS: we used a light scattering system to measure the transient volume changes of HL-1 cells when subjected to osmotic challenge. RESULTS: We found that HL-1 cells correct for their volume excess by undergoing regulatory volume decrease (RVD), and also respond to hypertonic stress with a regulatory volume increase (RVI). Rate of RVD was 0.08 ± 0.04 intensity/min, and rate of RVI was 0.09 ± 0.01 intensity/min. Volume recovery was 83.68 ± 5.73 % for RVD and 92.3 ± 2.3 % for RVI. Bumetanide 50 µM inhibited volume recovery, from 92.3 ± 2.3 % (control) to 24.6 ± 8.8 % and reduced the rate of RVI from 0.070 ± 0.020 intensity/min (control) to 0.010 ± 0.005 intensity/min. 50 µM DIDS reduced volume recovery to 42.93 ± 7.7 % and rate of RVI, to 0.03 ± 0.01 intensity/min. CONCLUSIONS: these results suggest that bumetanide- and DIDS-sensitive mechanisms are involved in the RVI of HL-1 cells, which points to the involvement of the Na(+)/K(+)/2Cl(-) cotransporter and Cl(-)/bicarbonate exchanger in RVI, respectively.

Galectin-1 controls cardiac inflammation and ventricular remodeling during acute myocardial infarction.

Galectin-1 (Gal-1), an evolutionarily conserved ß-galactoside-binding lectin, plays essential roles in the control of inflammation and neovascularization. Although identified as a major component of the contractile apparatus of cardiomyocytes, the potential role of Gal-1 in modulating heart pathophysiology is uncertain. Here, we aimed to characterize Gal-1 expression and function in the infarcted heart. Expression of Gal-1 was substantially increased in the mouse heart 7 days after acute myocardial infarction (AMI) and in hearts from patients with end-stage chronic heart failure. This lectin was localized mainly in cardiomyocytes and inflammatory infiltrates in peri-infarct areas, but not in remote areas. Both simulated hypoxia and proinflammatory cytokines selectively up-regulated Gal-1 expression in mouse cardiomyocytes, whereas anti-inflammatory cytokines inhibited expression of this lectin or had no considerable effect. Compared with their wild-type counterpart, Gal-1-deficient (Lgals1(-/-)) mice showed enhanced cardiac inflammation, characterized by increased numbers of macrophages, natural killer cells, and total T cells, but reduced frequency of regulatory T cells, leading to impaired cardiac function at baseline and impaired ventricular remodeling 7 days after nonreperfused AMI. Treatment of mice with recombinant Gal-1 attenuated cardiac damage in reperfused AMI. Taken together, our results indicate a protective role for Gal-1 in normal cardiac homeostasis and postinfarction remodeling by preventing cardiac inflammation. Thus, Gal-1 treatment represents a potential novel strategy to attenuate heart failure in AMI.

Interaction map of the Trypanosoma cruzi ribosomal P protein complex (stalk) and the elongation factor 2.

The large subunit of the eukaryotic ribosome possesses a long and protruding stalk formed by the ribosomal P proteins. This structure is involved in the translation step of protein synthesis through interaction with the elongation factor 2 (EF-2). The Trypanosoma cruzi stalk complex is composed of four proteins of about 11 kDa, TcP1α, TcP1ß, TcP2α, TcP2ß and a fifth TcP0 of about 34 kDa. In a previous work, a yeast two-hybrid (Y2H) protein-protein interaction map of T. cruzi ribosomal P proteins was generated. In order to gain new insight into the assembly of the stalk, a complete interaction map was generated by surface plasmon resonance (SPR) and the kinetics of each interaction was calculated. All previously detected interactions were confirmed and new interacting pairs were found, such as TcP1ß-TcP2α and TcP1ß-TcP2ß. Moreover P2 but not P1 proteins were able to homo-oligomerize. In addition, the region comprising amino acids 210-270 on TcP0 was identified as the region interacting with P1/P2 proteins, using Y2H and SPR. The interaction domains on TcP2ß were also mapped by SPR identifying two distinct regions. The assembly order of the pentameric complex was assessed by SPR showing the existence of a hierarchy in the association of the different P proteins forming the stalk. Finally, the TcEF-2 gene was identified, cloned, expressed and refolded. Using SPR analysis we showed that TcEF-2 bound with similar affinity to the four P1/P2 ribosomal P proteins of T. cruzi but with reduced affinity to TcP0.

The heavy chain variable segment gene repertoire in chronic Chagas' heart disease.

Patients chronically infected with Trypanosoma cruzi develop chronic Chagas' heart disease (cChHD). Their Ab response is suspected to be involved in the cardiac pathogenesis. Reactivity of serum Abs from these patients has been extensively studied but little is known about the diversity of the in vivo IgG repertoire. We analyzed 125 variable H chain (VH) genes and compared it to repertoires from healthy individuals, and patients with autoimmune processes and other infections. VH were from plasma cells isolated from heart tissue of three cChHD patients and from a Fab combinatorial library derived from bone marrow of another cChHD patient. The role of the parasite in shaping the Ab repertoire was assessed analyzing VH genes before and after panning against T. cruzi Ag. Among recovered VH genes, a significantly increased representation of VH4 was observed. Plasma cells at the site of cardiac infiltration showed an increased VH1 usage. CDR3 lengths were similar to the ones found in the healthy repertoire and significantly shorter than in other infections. VH derived from anti-T. cruzi Fab and plasma cells showed a higher proportion of hypermutated genes, 46.9% and 43.75%, respectively, vs 30.9% of the cChHD patient repertoire, pointing to the role of parasite Ags in the shaping of the humoral response in Chagas' disease. No histological evidence of germinal center-like structures was observed in heart tissue. In accordance, VH analysis of heart plasmocytes revealed no evidence of clonal B cell expansion, suggesting that they migrated into heart tissue from secondary lymphoid organs.

Human recombinant antibodies against Trypanosoma cruzi ribosomal P2ß protein.

Patients with chronic Chagas' Heart Disease (cChHD) develop an antibody response that is suspected to be involved in the cardiac pathogenesis. The response against Trypanosoma cruzi ribosomal P proteins is of particular interest, as these antibodies can cross-react with host cardiac receptors causing electrophysiological alterations. To better understand the humoral anti-P response we constructed a single-chain variable fragment library derived from a cChHD patient. The variable heavy and light regions were amplified from bone-marrow RNA and subcloned into the vector pComb3X. The phage library was subsequently panned against T. cruzi ribosomal P2ß protein (TcP2ß). We obtained 3 different human recombinant antibodies that specifically reacted with TcP2ß in ELISA and Western blots. Two of them reacted with the C-terminal region of TcP2ß, peptide R13, as the recombinant autoanti-P antibodies from Systemic Lupus Erythematosus (SLE) patients. Interestingly, the third one was specific for TcP2ß but did not recognize R13, confirming the specific nature of the anti-P response in Chagas disease. Neither sequence nor VH usage similarities between Chagas and SLE anti-P autoantibodies were observed. Herein, the first human mAbs against TcP2ß have been obtained and characterized showing that the humoral anti-P response is directed against the parasite and does not include an autoimmune component.

T Cell Specificity: A Great Challenge in Chagas Disease.

The CD4+ and CD8+ T cell immune response against T. cruzi, the parasite causing Chagas disease, are relevant for both parasite control and disease pathogenesis. Several studies have been focused on their phenotype and functionally, but only a few have drilled down to identify the parasite proteins that are processed and presented to these cells, especially to CD4+ T lymphocytes. Although approximately 10,000 proteins are encoded per haploid T. cruzi genome, fewer than 200 T cell epitopes from 49 T. cruzi proteins have been identified so far. In this context, a detailed knowledge of the specific targets of T cell memory response emerges as a prime tool for the conceptualization and development of prophylactic or therapeutic vaccines, an approach with great potential to prevent and treat this chronic disease. Here, we review the available information about this topic in a comprehensive manner and discuss the future challenges in the field.

Galectins in Chagas Disease: A Missing Link Between Trypanosoma cruzi Infection, Inflammation, and Tissue Damage.

Trypanosoma cruzi, the protozoan parasite causative agent of Chagas disease, affects about seven million people worldwide, representing a major global public health concern with relevant socioeconomic consequences, particularly in developing countries. In this review, we discuss the multiple roles of galectins, a family of ß-galactoside-binding proteins, in modulating both T. cruzi infection and immunoregulation. Specifically, we focus on galectin-driven circuits that link parasite invasion and inflammation and reprogram innate and adaptive immune responses. Understanding the dynamics of galectins and their ß-galactoside-specific ligands during the pathogenesis of T. cruzi infection and elucidating their roles in immunoregulation, inflammation, and tissue damage offer new rational opportunities for treating this devastating neglected disease.

Trypanosoma cruzi Induces Regulatory B Cell Alterations in Patients With Chronic Chagas Disease.

The clinical evolution of patients with chronic Chagas disease (CCD) is mainly associated with an excessive inflammation and a defective immunomodulatory profile caused by the interaction between T. cruzi and the host. Regulatory B (Breg) cells exert immune suppression mostly through IL-10 production (B10 cells), but also through IL-10-independent mechanisms. Previously, we demonstrated that CCD patients with cardiomyopathy show changes in the ex vivo Breg cell phenotypic distribution although maintain IL-10 production capacity. Here, we sought to identify potential alterations on Breg cells upon in vitro stimulation. Isolated B cells from CCD patients with or without cardiomyopathy and non-infected (NI) donors were stimulated with T. cruzi lysate or CpG + CD40L, and characterized by flow cytometry based on the expression of CD24, CD27, CD38, and the regulatory molecules IL-10 and PD-L1. IL-10 and IL-17 secretion in the supernatant of B cells was evaluated by ELISA. Data showed that T. cruzi stimulation diminished the expression of CD24 and CD38 on CD27- B cells while reducing the percentage of CD24high inside CD27+ B cells. Furthermore, T. cruzi induced a regulatory B cell phenotype by increasing B10 cells and IL-10 secretion in all the groups. The innate-like B10 cells expansion observed in patients with cardiomyopathy would be associated with CD27- B10 cell subsets, while no predominant phenotype was found in the other groups. Patients with cardiomyopathy also displayed higher IL-17 secretion levels in T. cruzi-activated B cells. CpG + CD40L stimulation revealed that B cells from CCD patients and NI donors had the same ability to differentiate into B10 cells and secrete IL-10 in vitro. Additionally, CCD patients showed an increased frequency of CD24-CD27- B cells and a reduction in the percentage of CD24highCD27+ Breg cells, which appeared to be inversely correlated with the presence of T. cruzi DNA in blood. Finally, CCD patients exhibited a higher frequency of PD-L1+ B cells in T. cruzi-stimulated samples, suggesting that IL-10-independent mechanisms could also be tangled in the control of inflammation. Altogether, our results provide evidence about the potential role of Breg cells in the immune response developed against T. cruzi and its contribution to chronic Chagas cardiomyopathy.

Ex vivo characterization of Breg cells in patients with chronic Chagas disease.

Despite the growing importance of the regulatory function of B cells in many infectious diseases, their immunosuppressive role remains elusive in chronic Chagas disease (CCD). Here, we studied the proportion of different B cell subsets and their capacity to secrete IL-10 ex vivo in peripheral blood from patients with or without CCD cardiomyopathy. First, we immunophenotyped peripheral blood mononuclear cells from patients according to the expression of markers CD19, CD24, CD38 and CD27 and we showed an expansion of total B cell and transitional CD24highCD38high B cell subsets in CCD patients with cardiac involvement compared to non-infected donors. Although no differences were observed in the frequency of total IL-10 producing B cells (B10) among the groups, CCD patients with cardiac involvement showed an increased proportion of naïve B10 cells and a tendency to a higher frequency of transitional B10 cells compared to non-infected donors. Our research demonstrates that transitional B cells are greatly expanded in patients with the cardiac form of CCD and these cells retain the ability to secrete IL-10. These findings provide insight into the phenotypic distribution of regulatory B cells in CCD, an important step towards new strategies to prevent cardiomyopathy associated with T. cruzi infection.

Interaction Between the Angiotensin-(1-7) Mas Receptor and the Dopamine D2 Receptor: Implications in Inflammation.

[Figure: see text].

Recombinant antibody against Trypanosoma cruzi from patients with chronic Chagas heart disease recognizes mammalian nervous system.

BACKGROUND: To deeply understand the role of antibodies in the context of Trypanosoma cruzi infection, we decided to characterize A2R1, a parasite antibody selected from single-chain variable fragment (scFv) phage display libraries constructed from B cells of chronic Chagas heart disease patients. METHODS: Immunoblot, ELISA, cytometry, immunofluorescence and immunohistochemical assays were used to characterize A2R1 reactivity. To identify the antibody target, we performed an immunoprecipitation and two-dimensional electrophoresis coupled to mass spectrometry and confirmed A2R1 specific interaction by producing the antigen in different expression systems. Based on these data, we carried out a comparative in silico analysis of the protein target´s orthologues, focusing mainly on post-translational modifications. FINDINGS: A2R1 recognizes a parasite protein of ~50 kDa present in all life cycle stages of T. cruzi, as well as in other members of the kinetoplastid family, showing a defined immunofluorescence labeling pattern consistent with the cytoskeleton. A2R1 binds to tubulin, but this interaction relies on its post-translational modifications. Interestingly, this antibody also targets mammalian tubulin only present in brain, staining in and around cell bodies of the human peripheral and central nervous system. INTERPRETATION: Our findings demonstrate for the first time the existence of a human antibody against T. cruzi tubulin capable of cross-reacting with a human neural protein. This work re-emphasizes the role of molecular mimicry between host and parasitic antigens in the development of pathological manifestations of T. cruzi infection.

A Single Dose of a Hybrid hAdV5-Based Anti-COVID-19 Vaccine Induces a Long-Lasting Immune Response and Broad Coverage against VOC.

Most approved vaccines against COVID-19 have to be administered in a prime/boost regimen. We engineered a novel vaccine based on a chimeric human adenovirus 5 (hAdV5) vector. The vaccine (named CoroVaxG.3) is based on three pillars: (i) high expression of Spike to enhance its immunodominance by using a potent promoter and an mRNA stabilizer; (ii) enhanced infection of muscle and dendritic cells by replacing the fiber knob domain of hAdV5 by hAdV3; (iii) use of Spike stabilized in a prefusion conformation. The transduction with CoroVaxG.3-expressing Spike (D614G) dramatically enhanced the Spike expression in human muscle cells, monocytes and dendritic cells compared to CoroVaxG.5 that expressed the native fiber knob domain. A single dose of CoroVaxG.3 induced a potent humoral immunity with a balanced Th1/Th2 ratio and potent T-cell immunity, both lasting for at least 5 months. Sera from CoroVaxG.3-vaccinated mice was able to neutralize pseudoviruses expressing B.1 (wild type D614G), B.1.117 (alpha), P.1 (gamma) and B.1.617.2 (delta) Spikes, as well as an authentic P.1 SARS-CoV-2 isolate. Neutralizing antibodies did not wane even after 5 months, making this kind of vaccine a likely candidate to enter clinical trials.

Angiotensin-(1-7) upregulates cardiac nitric oxide synthase in spontaneously hypertensive rats.

It has been shown that angiotensin (ANG)-(1-7) activates nitric oxide synthase (NOS) in isolated ventricular myocytes from normotensive rats. Since many ANG-(1-7) actions are enhanced in situations of increased ANG II activity, as in hypertension, in this study we investigated the in vivo effect of ANG-(1-7) on NOS activity and expression of endothelial (eNOS), neuronal (nNOS), and inducible NOS (iNOS) in ventricles from spontaneously hypertensive rats (SHR). Rats were subjected to a 60-min ANG-(1-7) infusion (0.35 nmol/min); controls received saline. NOS activity was measured using the NADPH diaphorase histochemical method and by the conversion of L-[(14)C]arginine to citrulline, and NOS phosphorylation and expression were determined using Western blotting. In SHR, ANG-(1-7) infusion diminished mean arterial pressure from 180 ± 9 to 146 ± 9 mmHg (P < 0.05), and this effect was prevented by nitro-l-arginine methyl ester (l-NAME), a NOS inhibitor. In addition, NOS activity and eNOS phosphorylation were increased by ANG-(1-7) infusion. Ventricular eNOS and nNOS expression were increased 67.4 ± 6.4 and 51 ± 10%, respectively, by ANG-(1-7), whereas iNOS was not changed. In another set of experiments, we evaluated the mechanism by which ANG-(1-7) modifies NOS activity. Isolated ventricle slices preincubated with ANG-(1-7) showed an increase in NOS activity and eNOS phosphorylation, which was blocked by an AT(2) and a bradykinin B(2) receptor antagonist, but not by the Mas receptor antagonist. Our results show that in rats in a hypertensive state, ANG-(1-7) infusion upregulates cardiac NOS expression and activity through an AT(2)- and bradykinin-dependent mechanism. In this way ANG-(1-7) may elicit its cardioprotective action and contribute to some of the counterregulatory AT(2) receptor effects that oppose the AT(1) receptor-mediated effects.