RESUMO

Bovine trypanosomosis, caused by Trypanosoma vivax, currently affects cattle and has a significant economic impact in sub-Saharan Africa and South America. The development of new diagnostic antigens is essential to improve and refine existing methods. Our study evaluated the efficacy of two recombinant antigens in detecting specific antibodies in cattle. These antigens are derivatives of an invariant surface glycoprotein (ISG) from T. vivax. A fraction of a previously described antigen (TvY486_0045500), designated TvISGAf, from an African strain was evaluated, and a new ISG antigen from an American isolate, TvISGAm, was identified. The two antigens were expressed as fusion proteins in Escherichia coli: TvISGAf was fused to the MBP-His-tag, and TvISGAm was obtained as a His-tag fused protein. An ELISA evaluation was conducted using these antigens on 149 positive and 63 negative bovine samples. The diagnostic performance was enhanced by the use of a combination of both antigens (referred to as TvISG-based ELISA), achieving a sensitivity of 89.6% and specificity of 93.8%. Following the validation of the TvISG-based ELISA, the seroprevalence of T. vivax infection in 892 field samples from cattle in the central region of Argentina was determined. The mean seroprevalence of T. vivax was 53%, with variation ranging from 21% to 69% among the six departments studied. These results support the use of the TvISG ELISA as a valuable serological tool for the detection and monitoring of T. vivax infection in cattle. Furthermore, we report for the first time the seroprevalence of T. vivax in Argentina, which highlights the widespread endemic nature of the disease in the region. In order to effectively manage the increasing spread of T. vivax in the vast livestock production areas of South America, it is essential to implement consistent surveillance programs and to adopt preventive strategies.

Assuntos

Antígenos de Protozoários , Doenças dos Bovinos , Ensaio de Imunoadsorção Enzimática , Testes Sorológicos , Trypanosoma vivax , Animais , Bovinos , Argentina/epidemiologia , Trypanosoma vivax/imunologia , Trypanosoma vivax/genética , Trypanosoma vivax/isolamento & purificação , Testes Sorológicos/métodos , Testes Sorológicos/veterinária , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/parasitologia , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/genética , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/veterinária , Anticorpos Antiprotozoários/sangue , Sensibilidade e Especificidade , Tripanossomíase Africana/diagnóstico , Tripanossomíase Africana/veterinária , Tripanossomíase Africana/epidemiologia , Gado/parasitologia

RESUMO

Myeloid-derived suppressor cells (MDSCs) comprise a heterogeneous population of myeloid origin and immature state, whose hallmark is the capacity to suppress T cells and other immune populations. In mice, the first approach to identify MDSCs relies in the measurement of their phenotypical markers: CD11b and GR-1. In addition, two main subtypes of MDSCs have been defined based on the expression of the following markers: CD11b+ Ly6G- Ly6C+ (monocytic-MDSCs, M-MDSCs) and CD11b+ Ly6G+ Ly6C+/low (polymorphonuclear-MDSCs, PMN-MDSCs). Since CD11b+ GR-1+ (Ly6C+/Ly6G+) MDSCs can increase significantly in peripheral blood during numerous acute or chronic processes, measuring alterations in the phenotypic markers CD11b and GR-1 could be important as a first step before assessing the suppressive function of the cells. In many cases it could be necessary to measure CD11b+ Gr-1+ cells from a minimum volume of peripheral blood cells without greatly affecting animal viability, since this approach would allow for further studies to be conducted on subsequent days, such as measuring parameters of the immune response or even survival in the context of the pathology under study. The following protocol describes a simple and optimized protocol for measuring the presence of CD11b+ GR-1+ (Ly6C+/Ly6G+) myeloid cells using 2+ channel flow cytometry, from a minimum volume of mouse peripheral blood obtained by facial vein puncture.

Assuntos

Monócitos , Células Mieloides , Camundongos , Animais , Células Mieloides/metabolismo , Linfócitos T , Citometria de Fluxo , Camundongos Endogâmicos C57BL

RESUMO

Trans-sialidase (TS) superfamily of proteins comprises eight subgroups, being the proteins of Group-I (TS-GI) promising immunogens in vaccine approaches against Trypanosoma cruzi. Strikingly, TS-GI antigenic variability among parasite lineages and their influence on vaccine development has not been previously analyzed. Here, a search in GenBank detects 49 TS-GI indexed sequences, whereas the main infecting human different parasite discrete typing units (DTU) are represented. In silico comparison among these sequences indicate that they share an identity above 92%. Moreover, the antigenic regions (T-cell and B-cell epitopes) are conserved in most sequences or present amino acid substitutions that scarcely may alter the antigenicity. Additionally, since the generic term TS is usually used to refer to different immunogens of this broad family, a further in silico analysis of the TS-GI-derived fragments tested in preclinical vaccines was done to determine the coverage and identity among them, showing that overall amino acid identity of vaccine immunogens is high, but the segment coverage varies widely. Accordingly, strong H-2K, H-2I, and B-cell epitopes are dissimilarly represented among vaccine TS-derived fragments depending on the extension of the TG-GI sequence used. Moreover, bioinformatic analysis detected a set of 150 T-cell strong epitopes among the DTU-indexed sequences that strongly bind human HLA-I supertypes. In all currently reported experimental vaccines based on TS-GI fragments, mapping these 150 epitopes showed that they are moderately represented. However, despite vaccine epitopes do not present all the substitutions observed in the DTUs, these regions of the proteins are equally recognized by the same HLAs.  Interestingly, the predictions regarding global and South American population coverage estimated in these 150 epitopes are similar to the estimations in experimental vaccines when the complete sequence of TS-GI is used as an antigen. In silico prediction also shows that a number of these MHC-I restricted T-cell strong epitopes could be also cross-recognized by HLA-I supertypes and H-2Kb or H-2Kd backgrounds, indicating that these mice may be used to improve and facilitate the development of new TS-based vaccines and suggesting an immunogenic and protective potential in humans. Further molecular docking analyses were performed to strengthen these results. Taken together, different strategies that would cover more or eventually fully of these T-cell and also B-cell epitopes to reach a high level of coverage are considered.

Assuntos

Trypanosoma cruzi , Camundongos , Humanos , Animais , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo , Epitopos de Linfócito B/genética , Simulação de Acoplamento Molecular , Glicoproteínas/metabolismo

RESUMO

During an infection the immune system produces pathogen-specific antibodies. These antibody repertoires become specific to the history of infections and represent a rich source of diagnostic markers. However, the specificities of these antibodies are mostly unknown. Here, using high-density peptide arrays we examined the human antibody repertoires of Chagas disease patients. Chagas disease is a neglected disease caused by Trypanosoma cruzi, a protozoan parasite that evades immune mediated elimination and mounts long-lasting chronic infections. We describe a proteome-wide search for antigens, characterised their linear epitopes, and show their reactivity on 71 individuals from diverse human populations. Using single-residue mutagenesis we revealed the core functional residues for 232 of these epitopes. Finally, we show the diagnostic performance of identified antigens on challenging samples. These datasets enable the study of the Chagas antibody repertoire at an unprecedented depth and granularity, while also providing a rich source of serological biomarkers.

Assuntos

Doença de Chagas , Trypanosoma cruzi , Humanos , Trypanosoma cruzi/genética , Epitopos , Especificidade de Anticorpos , Ensaio de Imunoadsorção Enzimática , Doença de Chagas/parasitologia , Antígenos de Protozoários/genética , Anticorpos , América , Anticorpos Antiprotozoários

RESUMO

Chagas disease (CD), caused by the protozoan parasite Trypanosoma cruzi, is the third largest parasitic disease burden globally. Currently, more than 6 million people are infected, mainly in Latin America, but international migration has turned CD into an emerging health problem in many nonendemic countries. Despite intense research, a vaccine is still not available. A complex parasite life cycle, together with numerous immune system manipulation strategies, may account for the lack of a prophylactic or therapeutic vaccine. There is substantial experimental evidence supporting that T. cruzi acute infection generates a strong immunosuppression state that involves numerous immune populations with regulatory/suppressive capacity. Myeloid-derived suppressor cells (MDSCs), Foxp3+ regulatory T cells (Tregs), regulatory dendritic cells and B regulatory cells are some of the regulatory populations that have been involved in the acute immune response elicited by the parasite. The fact that, during acute infection, MDSCs increase notably in several organs, such as spleen, liver and heart, together with the observation that depletion of those cells can decrease mouse survival to 0%, strongly suggests that MDSCs play a major role during acute T. cruzi infection. Accumulating evidence gained in different settings supports the capacity of MDSCs to interact with cells from both the effector and the regulatory arms of the immune system, shaping the outcome of the response in a very wide range of scenarios that include pathological and physiological processes. In this sense, the aim of the present review is to describe the main knowledge about MDSCs acquired so far, including several crosstalk with other immune populations, which could be useful to gain insight into their role during T. cruzi infection.

Assuntos

Doença de Chagas , Células Supressoras Mieloides , Trypanosoma cruzi , Animais , Camundongos , Doença de Chagas/parasitologia , Doença de Chagas/prevenção & controle , Sistema Imunitário , Linfócitos T Reguladores

RESUMO

Trypanosoma cruzi, the agent of Chagas disease, can infect through conjunctive or oral mucosas. Therefore, the induction of mucosal immunity by vaccination is relevant not only to trigger local protection but also to stimulate both humoral and cell-mediated responses in systemic sites to control parasite dissemination. In a previous study, we demonstrated that a nasal vaccine based on a Trans-sialidase (TS) fragment plus the mucosal STING agonist c-di-AMP, was highly immunogenic and elicited prophylactic capacity. However, the immune profile induced by TS-based nasal vaccines at the nasopharyngeal-associated lymphoid tissue (NALT), the target site of nasal immunization, remains unknown. Hence, we analyzed the NALT cytokine expression generated by a TS-based vaccine plus c-di-AMP (TSdA+c-di-AMP) and their association with mucosal and systemic immunogenicity. The vaccine was administered intranasally, in 3 doses separated by 15 days each other. Control groups received TSdA, c-di-AMP, or the vehicle in a similar schedule. We demonstrated that female BALB/c mice immunized intranasally with TSdA+c-di-AMP boosted NALT expression of IFN-γ and IL-6, as well as IFN-ß and TGF-ß. TSdA+c-di-AMP increased TSdA-specific IgA secretion in the nasal passages and also in the distal intestinal mucosa. Moreover, T and B-lymphocytes from NALT-draining cervical lymph nodes and spleen showed an intense proliferation after ex-vivo stimulation with TSdA. Intranasal administration of TSdA+c-di-AMP provokes an enhancement of TSdA-specific IgG2a and IgG1 plasma antibodies, accompanied by an increase IgG2a/IgG1 ratio, indicative of a Th1-biased profile. In addition, immune plasma derived from TSdA+c-di-AMP vaccinated mice exhibit in-vivo and ex-vivo protective capacity. Lastly, TSdA+c-di-AMP nasal vaccine also promotes intense footpad swelling after local TSdA challenge. Our data support that TSdA+c-di-AMP nasal vaccine triggers a NALT mixed pattern of cytokines that were clearly associated with an evident mucosal and systemic immunogenicity. These data are useful for further understanding the immune responses elicited by the NALT following intranasal immunization and the rational design of TS-based vaccination strategies for prophylaxis against T. cruzi.

Assuntos

Doença de Chagas , Trypanosoma cruzi , Vacinas , Feminino , Animais , Camundongos , Administração Intranasal , Imunidade nas Mucosas , Linfonodos , Doença de Chagas/prevenção & controle , Citocinas/metabolismo , Nasofaringe/metabolismo , Mucosa Intestinal/metabolismo , Imunoglobulina G , Camundongos Endogâmicos BALB C

RESUMO

Low-cost adjuvants are urgently needed for the development of veterinary vaccines able to trigger strong immune responses. In this work, we describe a method to obtain a low-cost cage-like particles (ISCOMATRIX-like) adjuvant useful to formulate veterinary vaccines candidates. The main components to form the particles are lipids and saponins, which were obtained from egg yolk by ethanolic extraction and by dialyzing a non-refined saponins extract, respectively. Lipids were fully characterized by thin layer chromatography (TLC) and gas-chromatography (GC) and enzymatic methods, and saponins were characterized by TLC, HPLC and MALDI-TOF. Cage-like particles were prepared with these components or with commercial inputs. Both particles and the traditional Alum used in veterinary vaccines were compared by immunizing mice with Ovalbumin (OVA) formulated with these adjuvants and assessing IgG1, IgG2a anti OVA antibodies and specific Delayed-type Hypersensitivity (DTH). In the yolk extract, a mixture of phospholipids, cholesterol and minor components of the extract (e.g. lyso-phospholipids) with suitable proportions to generate cage-like particles was obtained. Also, semi-purified saponins with similar features to those of the QuilA® were obtained. Cage-like particles prepared with these components have 40-50 nm and triggers similar levels of Anti-OVA IgG1 and DTH than with commercial inputs but higher specific-IgG2a. Both adjuvants largely increased the levels of IgG1, IgG2a and DTH in relation to the formulation with Alum. The methods described to extract lipids from egg yolk and saponins from non-refined extract allowed us to obtain an inexpensive and highly effective adjuvant.

Assuntos

Saponinas , Vacinas , Adjuvantes Imunológicos/química , Animais , Imunoglobulina G , Camundongos , Ovalbumina

RESUMO

Transcutaneous immunization (TCI) provides a valuable alternative approach to conventional vaccination because of the high accessibility and the exceptional immunological characteristics of the skin, but its application is limited by the low permeability of the stratum corneum. Although nanogels (NGs) have proven to enhance skin penetration of macromolecules with minimum damage, their use in TCI remains almost unexplored. In this context, this article evaluates the performance of novel film-forming NGs (FF-NGs) as TCI. This TCI platform consists of NGs with multilobular morphology that positively combines the properties of cross-linked poly(N-vinylcaprolactam), like thermoresponsiveness and the ability to load and release a cargo, with the film-forming capacity of low Tg lobes. FF-NGs and formed films are characterized at different levels. Formed films show to be able to uniformly load an antigenic protein and release it with a profile depending on the temperature and on their FF-NGs content. In vivo studies have demonstrated that FF-NGs promote the penetration of not only an antigenic protein, but also an adjuvant until the immunocompetent area of skin, generating an adjuvant-dependent specific immune response. Finally, this study provides a successful proof of concept that FF-NGs can be a powerful tool for the transcutaneous release of complex formulations.

Assuntos

Pele , Vacinação , Administração Cutânea , Antígenos , Imunização , Nanogéis , Pele/metabolismo

RESUMO

The new generation of vaccines for Chagas disease, are focused to induce both humoral and cellular response to effectively control Trypanosoma cruzi parasites. The administration of vaccine formulations intranasally has the advantage over parenteral routes that can induce a specific response at mucosal and systemic levels. This study aimed to evaluate and compare the immunogenicity and prophylactic effectiveness of two Trans-sialidase (TS)-based mucosal vaccines against T. cruzi administered intranasally. Vaccines consisted of a recombinant fragment of TS expressed in Lactococcus lactis formulated in two different adjuvants. The first, was an immunostimulant particle (ISPA, an ISCOMATRIX-like adjuvant), while the second was the dinucleotide c-di-AMP, which have shown immunostimulant properties at the mucosal level. BALB/c mice were immunized intranasally (3 doses, one every two weeks) with each formulation (TS + ISPA or TS + c-di-AMP) and with TS alone or vehicle (saline solution) as controls. Fifteen days after the last immunization, both TS + ISPA or TS + c-di-AMP induced an evident systemic humoral and cellular response, as judged by the increased plasma anti-TS IgG2a titers and IgG2a/IgG1 ratio and enhanced cellular response against TS. Plasma derived antibodies from TS + c-di-AMP also inhibit in vitro the invasion capacity of T. cruzi. Furthermore, specific secretory IgA was more enhanced in TS + c-di-AMP group. Protective efficacy was proved in vaccinated animals by an oral T. cruzi-challenge. Parasitemia control was only achieved by animals vaccinated with TS + c-di-AMP, despite all vaccinates groups showed enhanced CD8+IFN-γ+ T cell numbers. In addition, it was reflected during the acute phase in a significant reduction of tissue parasite load, clinical manifestations and diminished tissue damage. The better prophylactic capacity elicited by TS + c-di-AMP was related to the induction of neutralizing plasma antibodies and augmented levels of mucosal IgA since TS + ISPA and TS + c-di-AMP groups displayed similar immunogenicity and CD8+IFN-γ+ T-cell response. Therefore, TS + c-di-AMP formulation appears as a promising strategy for prophylaxis against T. cruzi.

Assuntos

Doença de Chagas , Vacinas Protozoárias , Trypanosoma cruzi , Animais , Doença de Chagas/prevenção & controle , Fosfatos de Dinucleosídeos , Glicoproteínas , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Neuraminidase

RESUMO

The difficulties encountered in achieving treatments for chronic Chagas disease have promoted the investigation of new therapeutic strategies. In this study, we used two murine models of Trypanosoma cruzi chronic infection to determine the usefulness of applying a therapeutic vaccine alone or followed by benznidazole (Bz) chemotherapy. A vaccine formulation based on an N-terminal fragment of Trans-sialidase (TS) and Immunostimulant Particle Adjuvant (ISPA) - TSNt-ISPA was obtained. Firstly, the immunogenicity and protective capacity of TSNt-ISPA was demonstrated as a prophylactic formulation in an acute model of infection. Later, the formulation was assessed as a therapeutic vaccine alone or combined with (Bz) using two models of chronic infection. BALB/c mice chronically infected with Sylvio X10/4 or Tulahuen cl2 T. cruzi strains were not treated as control or treated only with the therapeutic vaccine TSNt-ISPA, with a combined treatment TSNt-ISPA+Bz (Bz applied after the vaccine), or only with Bz. The vaccination schedule consisted of TSNt-ISPA administration at days110, 120, and 130 post-infection (pi) and Bz administration was performed daily from day 140 to 170 pi. At day 273 pi, electrocardiographic (ECG) parameters, heart parasite load, myocarditis, and heart fibrosis were assessed. In both models, therapeutic administration of TSNt-ISPA reduced ECG alterations and the cardiac tissue damage observed in the chronic phase. Moreover, vaccine treatment significantly decreased heart parasite load in both Sylvio X10/4 and Tulahuen cl2 infected mice. The combined treatment, but not Bz or vaccine administration alone, allowed to restore ECG parameters in Tulahuen cl2 infected mice. The results indicate the usefulness of the therapeutic TSNt-ISPA formulation in BALB/c mice chronically infected with Sylvio X10/4 or Tulahuen cl2 strain. For the mice infected with T. cruzi Tulahuen cl2 strain, the combined treatment with the vaccine and Bz had a more positive effect on the course of heart disease than the individual treatments with the vaccine or Bz alone.

Assuntos

Doença de Chagas , Nitroimidazóis , Tripanossomicidas , Trypanosoma cruzi , Vacinas , Animais , Doença de Chagas/parasitologia , Camundongos , Nitroimidazóis/uso terapêutico , Infecção Persistente , Tripanossomicidas/uso terapêutico , Vacinas/uso terapêutico

RESUMO

Trypanosoma cruzi (T. cruzi) is a hemoflagellate protozoan parasite that causes Chagas disease, a neglected tropical disease that affects more than 6 million people around the world, mostly in Latin America. Despite intensive research, there is no vaccine available; therefore, new approaches are needed to further improve vaccine efficacy. It is well established that experimental T. cruzi infection induces a marked immunosuppressed state, which includes notably increases of CD11b+ GR-1+ myeloid-derived suppressor cells (MDSCs) in the spleen, liver and heart of infected mice. We previously showed that a trans-sialidase based vaccine (TSf-ISPA) is able to confer protection against a virulent T. cruzi strain, stimulating the effector immune response and decreasing CD11b+ GR-1+ splenocytes significantly. Here, we show that even in the immunological context elicited by the TSf-ISPA vaccine, the remaining MDSCs are still able to influence several immune populations. Depletion of MDSCs with 5 fluorouracil (5FU) at day 15 post-infection notably reshaped the immune response, as evidenced by flow cytometry of spleen cells obtained from mice after 21 days post-infection. After infection, TSf-ISPA-vaccinated and 5FU-treated mice showed a marked increase of the CD8 response, which included an increased expression of CD107a and CD44 markers in CD8+ cultured splenocytes. In addition, vaccinated and MDSC depleted mice showed an increase in the percentage and number of CD4+ Foxp3+ regulatory T cells (Tregs) as well as in the expression of Foxp3+ in CD4+ splenocytes. Furthermore, depletion of MDSCs also caused changes in the percentage and number of CD11chigh CD8α+ dendritic cells as well as in activation/maturation markers such as CD80, CD40 and MHCII. Thus, the obtained results suggest that MDSCs not only play a role suppressing the effector response during T. cruzi infection, but also strongly modulate the immune response in vaccinated mice, even when the vaccine formulation has a significant protective capacity. Although MDSC depletion at day 15 post-infection did not ameliorated survival or parasitemia levels, depletion of MDSCs during the first week of infection caused a beneficial trend in parasitemia and mice survival of vaccinated mice, supporting the possibility to target MDSCs from different approaches to enhance vaccine efficacy. Finally, since we previously showed that TSf-ISPA immunization causes a slight but significant increase of CD11b+ GR-1+ splenocytes, here we also targeted those cells at the stage of immunization, prior to T. cruzi challenge. Notably, 5FU administration before each dose of TSf-ISPA vaccine was able to significantly ameliorate survival and decrease parasitemia levels of TSf-ISPA-vaccinated and infected mice. Overall, this work supports that targeting MDSCs may be a valuable tool during vaccine design against T. cruzi, and likely for other pathologies that are characterized by the subversion of the immune system.

Assuntos

Doença de Chagas , Células Supressoras Mieloides , Vacinas Protozoárias , Trypanosoma cruzi , Animais , Doença de Chagas/prevenção & controle , Glicoproteínas , Camundongos , Neuraminidase

RESUMO

Potential activation of ß2 adrenergic receptors (ß2AR) by specific autoreactive antibodies (Abs) that arise during the host reaction to Trypanosoma cruzi, could contribute to the elevated prevalence of metabolic disturbances described in patients with chronic Chagas disease (CCD). This study aimed to determine the prevalence of anti-ß2AR Abs in patients with CCD, as well as the correlation of these Abs with the presence of glucose and lipid metabolism disturbances, in order to explore their association with an insulin resistance profile. Additionally, we tested the functional effects of anti-ß2AR Abs employing an in vitro bioassay with neuroendocrine cells expressing ß2AR. A clinical and metabolic evaluation including an OGTT was performed in 80 CCD patients and 40 controls. Anti-ß2AR Abs were measured by an in-house-developed ELISA, and the ß2 adrenergic activity of affinity-purified IgG fractions from patient' sera were assayed in CRE-Luc and POMCLuc transfected AtT-20 cells. A higher proportion of dysglycemia (72.5% vs. 37.5%; p = 0.001) was observed in the CCD group, accompanied by increased HOMA2-IR (p = 0.019), especially in subjects with Abs (+). Anti-ß2AR Abs reactivity (7.01 (2.39-20.5); p = 0.0004) and age >50 years (3.83 (1.30-11.25); p = 0.014) resulted as relevant for IR prediction (AUC: 0.786). Concordantly, Abs (+) CCD patients showed elevated metabolic risk scores and an increased prevalence of atherogenic dyslipidemia (p = 0.040), as compared to Abs (-) patients and controls. On functional bioassays, Abs exerted specific and dose-dependent ß2-agonist effects. Our findings suggest that anti-ß2AR Abs may induce the activation of ß2AR in other tissues besides the heart; furthermore, we show that in patients with CCD these Abs are associated with an insulin resistance profile and atherogenic dyslipidemia, providing biological plausibility to the hypothesis that adrenergic activation by anti-ß2AR Abs could contribute to the pathogenesis of metabolic disturbances described in CCD patients, increasing their cardiovascular risk.

RESUMO

The immunodominant B13 protein of Trypanosoma cruzi is found on the surface of trypomastigotes and exhibits cross-reactivity with the human cardiac myosin heavy chain; for which antibodies against this parasitic antigen may be involved in the development of disease pathology. In a cohort of chronically T. cruzi-infected adults, undergoing trypanocidal treatment, or not, we, therefore, decided to evaluate the levels of anti-B13 antibodies (ELISA-B13) and its eventual relationship with heart complaints. Two hundred twenty-eight serum samples from 76 chronically infected adults with an average follow-up of 24 years were analyzed. Thirty of them had received trypanocidal treatment. Among treated patients, anti-B13 Ab levels in successive samples showed a significant decrease in reactivity as the years after treatment increased (ANOVA test, p = 0.0049). At the end of the follow-up, 36.7% became non-reactive for ELISA B13. Untreated patients did not have significant variations in the level of anti-B13 antibodies during follow-up. None of the treated patients had electrocardiographic changes compatible with chronic chagasic cardiomyopathy, whereas 21.7% of those undergoing no treatment did show such kind of pathological electrocardiogram tracings. ELISA-B13 was reactive in all cases with heart involvement. Among untreated patients, there were no significant differences in anti-B13 antibodies when comparing individuals without proven pathology with those with chronic chagasic cardiomyopathy. Although treatment with trypanocidal drugs was followed by decreased anti-B13 antibody levels, such assessment was unhelpful in differentiating the evolution of chronic chagasic heart disease.

Assuntos

Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Doença de Chagas/tratamento farmacológico , Doença de Chagas/imunologia , Tripanossomicidas/uso terapêutico , Adulto , Animais , Argentina , Doença Crônica , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Feminino , Seguimentos , Humanos , Masculino , Nifurtimox/uso terapêutico , Nitroimidazóis/uso terapêutico , Estudos Retrospectivos , Trypanosoma cruzi , Adulto Jovem

RESUMO

BACKGROUND: It has been described that Trypanosoma cruzi is capable of promoting metabolic disturbances currently considered as cardiovascular risk factors. Moreover, it has been observed that the protozoa can remain in adipose tissue and alter its immune endocrine functions. The aim of this study was to characterize the thickness of epicardial adipose tissue (EAT) in patients with chronic Chagas disease (CCD) concerning their cardiovascular metabolic risk profile compared with those without CCD. METHODS: A cross-sectional study was performed including T. cruzi seropositive individuals categorized according to a standard CCD classification and a matched seronegative control group. Complete clinical examination, metabolic laboratory tests and transthoracic echocardiography to assess cardiac function and to quantify EAT were performed. RESULTS: Fifty-five individuals aged 46.7±11.9 y, 34 with CCD and 21 in the control group, were included. The CCD group presented higher EAT thickness in relation to controls (4.54±1.28 vs 3.22±0.99 mm; p=0.001), which was significantly associated with the presence of insulin resistance (OR=3, 95% CI 1.58 to 5.73; p<0.001). This group presented lower levels of plasmatic adiponectin than controls, especially in those patients with EAT ≥4.5 mm (p=0.005) who also presented with heart failure more frequently (p=0.01). CONCLUSION: In patients with CCD, a higher EAT thickness is observed and is associated with an increased metabolic risk profile indicated mainly by insulin resistance.

Assuntos

Doença de Chagas , Insuficiência Cardíaca , Tecido Adiposo/diagnóstico por imagem , Doença de Chagas/complicações , Estudos Transversais , Insuficiência Cardíaca/diagnóstico por imagem , Insuficiência Cardíaca/etiologia , Humanos , Pericárdio/diagnóstico por imagem

RESUMO

Foot-and-Mouth Disease (FMD) is an acute viral disease that causes important economy losses. Vaccines with new low-cost adjuvants that stimulate protective immune responses are needed and can be assayed in a mouse model to predict their effectiveness in cattle. Immunostimulant Particle Adjuvant (ISPA), also known as cage-like particle adjuvant, consisting of lipid boxes of dipalmitoyl-phosphatidylcholine, cholesterol, sterylamine, alpha-tocopherol, and QuilA saponin, was shown to enhance protection of a recombinant vaccine against Trypanosoma cruzi in a mouse model. Thus, in the present work, we studied the effects on the magnitude and type of immunity elicited in mice and cattle in response to a vaccine based on inactivated FMD virus (iFMDV) formulated with ISPA. It was demonstrated that iFMDV-ISPA induced protection in mice against challenge and elicited a specific antibody response in sera, characterized by a balanced Th1/Th2 profile. In cattle, the antibody titers reached corresponded to an expected percentage of protection (EPP) higher than 80%. EPP calculates the probability that livestock would be protected against a 10,000 bovine infectious doses challenge after vaccination. Moreover, in comparison with the non-adjuvanted iFMDV vaccine, iFMDV-ISPA elicited an increased specific T-cell response against the virus, including higher interferon gamma (IFNγ)+/CD8+ lymphocyte production in cattle. In this work, we report for first time that an inactivated FMDV serotype A vaccine adjuvanted with ISPA is capable of inducing protection against challenge in a murine model and of improving the specific immune responses against the virus in cattle.

RESUMO

Lactococcus lactis is a promising candidate for the development of mucosal vaccines. More than 20 years of experimental research supports this immunization approach. In addition, 3' 5'- cyclic di-adenosine monophosphate (c-di-AMP) is a bacterial second messenger that plays a key role in the regulation of diverse physiological functions (potassium and cellular wall homeostasis, among others). Moreover, recent studies showed that c-di-AMP has a strong mucosal adjuvant activity that promotes both humoral and cellular immune responses. In this study, we report the development of a novel mucosal vaccine prototype based on a genetically engineered L. lactis strain. First, we demonstrate that homologous expression of cdaA gen in L. lactis is able to increase c-di-AMP levels. Thus, we hypothesized that in vivo synthesis of the adjuvant can be combined with production of an antigen of interest in a separate form or jointly in the same strain. Therefore, a specifically designed fragment of the trans-sialidase (TScf) enzyme from the Trypanosoma cruzi parasite, the etiological agent of Chagas disease, was selected to evaluate as proof of concept the immune response triggered by our vaccine prototypes. Consequently, we found that oral administration of a L. lactis strain expressing antigenic TScf combined with another L. lactis strain producing the adjuvant c-di-AMP could elicit a TS-specific immune response. Also, an additional L. lactis strain containing a single plasmid with both cdaA and tscf genes under the Pcit and Pnis promoters, respectively, was also able to elicit a specific immune response. Thus, the current report is the first one to describe an engineered L. lactis strain that simultaneously synthesizes the adjuvant c-di-AMP as well as a heterologous antigen in order to develop a simple and economical system for the formulation of vaccine prototypes using a food grade lactic acid bacterium.

RESUMO

Staphylococcus aureus produces capsular polysaccharides (CPs) both in vivo and under defined culture conditions being serotypes 5 and 8 the most prevalent. S. aureus isolates that fail to produce CP5 or CP8 are defined as non-typeable (NT). Loss of capsule expression, however, may lead to S. aureus persistence in a chronically infected host. The prevalence of NT strains of S. aureus isolated from bovine mastitis varies according to the geographic origin of the strain. The aims of this work were to detect phenotypically and genotypically the capsular profile of 144 S. aureus isolated from bovine mastitis in Argentina, Chile, and Uruguay and explore the factors that are considered to be associated with capsule expression as presence of IS257, IScap, and agr typing of non-related collection. The detection of the IS257, IScap, cap genes, and agr typing was performed using PCR. The detection and quantification of capsular polysaccharide production were performed by ELISA assays. We found that 96% of the S. aureus isolates investigated carried cap5(8) genes but over 75% of strains do not express capsule in the three countries studied. However, only 6 isolates from Argentina carried the IScap element that totally suppressed the expression of the capsule, suggesting that other factors could influence on CP expression. Moreover, the agrI/NT association was statistically significant suggesting that this profile is a phenomenon observed not only in other parts of the world but also in our region.

Assuntos

Cápsulas Bacterianas/genética , Mastite Bovina/microbiologia , Polissacarídeos Bacterianos/genética , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/genética , Animais , Argentina , Bovinos , Chile , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , Indústria de Laticínios , Feminino , Genes Bacterianos , Sorogrupo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/metabolismo , Uruguai

Assuntos

Anticorpos Antiprotozoários/imunologia , Afinidade de Anticorpos , Imunoglobulina G/imunologia , Complicações Neoplásicas na Gravidez/diagnóstico , Toxoplasmose/diagnóstico , Técnicas de Laboratório Clínico/métodos , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Gravidez , Proteínas de Protozoários/imunologia , Proteínas Recombinantes/imunologia , Toxoplasma/imunologia

RESUMO

The use of chimeric molecules fusing several antigenic determinants is a promising strategy for the development of low-cost, standardized and reliable kits to determine specific antibodies. In this study, we designed and assessed a novel recombinant chimera that complements the performance of our previously developed chimera, CP1 [FRA and SAPA antigens (Ags)], to diagnose chronic Chagas disease. The new chimeric protein, named CP3, is composed of MAP, TcD and TSSAII/V/VI antigenic determinants. We compared the performance of both chimeric Ags using a panel of 67 Trypanosoma cruzi-reactive sera and 67 non-reactive ones. The sensitivity of CP3 vs CP1 was 100 and 90.2%, and specificity was 92.5 and 100%, respectively. The mixture of CP1 + CP3 achieved 100% of sensitivity and specificity. More importantly, an additional subset of 17 sera from patients with discordant results of conventional serological methods was analysed; the CP1 + CP3 mixture allowed us to accurately classify 14 of them with respect to IIF, the usual technique used in most of the reference centres. These results show an improved performance of the CP1 + CP3 mixture in comparison with enzyme-linked immunosorbent assay and indirect haemagglutination commercial assays.

Assuntos

Doença de Chagas/diagnóstico , Epitopos/imunologia , Trypanosoma cruzi/imunologia , Sequência de Aminoácidos , Doença de Chagas/parasitologia , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoensaio , Dados de Sequência Molecular , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade

RESUMO

Prophylactic and/or therapeutic vaccines have an important potential to control Trypanosoma cruzi (T. cruzi)infection. The involvement of regulatory/suppressor immune cells after an immunization treatment and T. cruzi infection has never been addressed. Here we show that a new trans-sialidase-based immunogen (TSf) was able to confer protection, correlating not only with beneficial changes in effector immune parameters, but also influencing populations of cells related to immune control. Regarding the effector response, mice immunized with TSf showed a TS-specific antibody response, significant delayed-type hypersensitivity (DTH) reactivity and increased production of IFN-γ by CD8+ splenocytes. After a challenge with T. cruzi, TSf-immunized mice showed 90% survival and low parasitemia as compared with 40% survival and high parasitemia in PBS-immunized mice. In relation to the regulatory/suppressor arm of the immune system, after T. cruzi infection TSf-immunized mice showed an increase in spleen CD4+ Foxp3+ regulatory T cells (Treg) as compared to PBS-inoculated and infected mice. Moreover, although T. cruzi infection elicited a notable increase in myeloid derived suppressor cells (MDSC) in the spleen of PBS-inoculated mice, TSf-immunized mice showed a significantly lower increase of MDSC. Results presented herein highlight the need of studying the immune response as a whole when a vaccine candidate is rationally tested.