RESUMO
American Tegumentary Leishmaniasis (ATL) is a disease of high severity and incidence in Brazil, in addition to being a worldwide concern in public health. Leishmania amazonensis is one of the etiological agents of ATL, and the inefficiency of control measures, associated with the high toxicity of the treatment and the lack of effective immunoprophylactic strategies, makes the development of vaccines indispensable and imminent. In this light, the present study proposes to elaborate a chimeric protein (rChiP), based on the fusion of multiple epitopes of CD4+/CD8+ T cells, identified in the immunoproteome of the parasites L. amazonensis and L. braziliensis. The designed chimeric protein was tested in the L. amazonensis murine model of infection using the following formulations: 25 µg of the rChiP in saline (rChiP group) and 25 µg of the rChiP plus 25 µg of MPLA-PHAD® (rChiP+MPLA group). After completing immunization, CD4+ and CD8+ T cells, stimulated with SLa-Antigen or rChiP, showed an increased production of nitric oxide and intracytoplasmic pro-inflammatory cytokines, in addition to the generation of central and effector memory T cells. rChiP and rChiP+MPLA formulations were able to promote an effective protection against L. amazonensis infection determined by a reduction in the development of skin lesions and lower parasitic burden. Reduction in the development of skin lesions and lower parasitic burden in the vaccinated groups were associated with an increase of nitrite, CD4+/CD8+IFN-γ+TNF-α+ and CD4+/CD8+CD44highCD62Lhigh/low T cells, IgGTotal, IgG2a, and lower rates of IgG1 and CD4+/CD8+IL-10+. This data suggests that proposed formulations could be considered potential tools to prevent ATL.
Assuntos
Adjuvantes Imunológicos , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Epitopos de Linfócito T , Memória Imunológica , Vacinas contra Leishmaniose , Leishmaniose Cutânea , Animais , Leishmaniose Cutânea/prevenção & controle , Leishmaniose Cutânea/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD4-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Camundongos , Vacinas contra Leishmaniose/imunologia , Feminino , Adjuvantes Imunológicos/administração & dosagem , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/genética , Leishmania braziliensis/imunologia , Lipídeo A/análogos & derivados , Lipídeo A/imunologia , Anticorpos Antiprotozoários/imunologia , Citocinas/metabolismo , Citocinas/imunologia , Modelos Animais de Doenças , Antígenos de Protozoários/imunologiaRESUMO
The diagnosis of tegumentary leishmaniasis (TL) is hampered by variable sensitivity and/or specificity of the tests. Serological assays are suitable to diagnose visceral leishmaniasis (VL); however, they present low performance for the detection of TL cases. Additionally, blood collection to obtain patient serum represents a challenge, as it is an invasive and uncomfortable procedure, requiring laboratorial infrastructure and trained professionals. In this context, the present study proposed to evaluate patient urine to detect TL, given that this analyte has proven to be effective in ELISA experiments for the detection of VL cases. For this, a Leishmania protein called LiHyV, two specific B-cell epitopes derived from protein amino acid sequence, and a Leishmania antigenic extract (SLA) were used as antigens. A total of 215 paired urine and serum samples were evaluated, and results showed that, when serum was employed as an analyte, rLiHyV, Peptide1, Peptide2, and SLA presented a sensitivity of 85 %, 29 %, 58 %, and 31 %, respectively, and a specificity of 97.5 %, 98 %, 100 %, and 97.5 %, respectively, in the diagnosis of TL. When urine was used, rLiHyV, Peptide1, Peptide2, and SLA presented a sensitivity of 95 %, 74 %, 67 %, and 52 %, respectively, and a specificity of 100 %, 99 %, 98 %, and 86 %, respectively. In conclusion, preliminary data suggest that urine could be considered as an alternative biological sample for the detection of TL cases.
Assuntos
Anticorpos Antiprotozoários , Antígenos de Protozoários , Ensaio de Imunoadsorção Enzimática , Leishmania , Leishmaniose Cutânea , Proteínas de Protozoários , Proteínas Recombinantes , Sensibilidade e Especificidade , Humanos , Ensaio de Imunoadsorção Enzimática/métodos , Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/urina , Proteínas de Protozoários/urina , Proteínas de Protozoários/imunologia , Antígenos de Protozoários/urina , Antígenos de Protozoários/imunologia , Leishmania/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/urina , Adulto , Feminino , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/urina , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Adolescente , Idoso , Urina/química , Urina/parasitologia , Criança , Pré-Escolar , Epitopos de Linfócito B/imunologiaRESUMO
BACKGROUND: Chagas disease (CD), a neglected parasitic disease caused by Trypanosoma cruzi, poses a significant health threat in Latin America and has emerged globally because of human migration. Trypanosoma cruzi infects humans and over 100 other mammalian species, including dogs, which are important sentinels for assessing the risk of human infection. Nonetheless, the serodiagnosis of T. cruzi in dogs is still impaired by the absence of commercial tests. In this study, we investigated the diagnostic accuracy of four chimeric recombinant T. cruzi IBMP antigens (IBMP-8.1, IBMP-8.2, IBMP-8.3, and IBMP-8.4) for detecting anti-T. cruzi antibodies in dogs, using latent class analysis (LCA). METHODS: We examined 663 canine serum samples, employing indirect ELISA with the chimeric antigens. LCA was utilized to establish a latent variable as a gold standard for T. cruzi infection, revealing distinct response patterns for each antigen. RESULTS: The IBMP (Portuguese acronym for the Molecular Biology Institute of Paraná) antigens achieved area under the ROC curve (AUC) values ranging from 90.9% to 97.3%. The highest sensitivity was attributed to IBMP-8.2 (89.8%), while IBMP-8.1, IBMP-8.3, and IBMP-8.4 achieved 73.5%, 79.6%, and 85.7%, respectively. The highest specificity was observed for IBMP-8.4 (98.6%), followed by IBMP-8.2, IBMP-8.3, and IBMP-8.1 with specificities of 98.3%, 94.4%, and 92.7%, respectively. Predictive values varied according to prevalence, indicating higher effectiveness in endemic settings. CONCLUSIONS: Our findings underscore the remarkable diagnostic performance of IBMP-8.2 and IBMP-8.4 for the serodiagnosis of Trypanosoma cruzi in dogs, representing a promising tool for the diagnosis of CD in dogs. These chimeric recombinant antigens may not only enhance CD surveillance strategies but also hold broader implications for public health, contributing to the global fight against this neglected tropical disease.
Assuntos
Anticorpos Antiprotozoários , Antígenos de Protozoários , Doença de Chagas , Doenças do Cão , Ensaio de Imunoadsorção Enzimática , Sensibilidade e Especificidade , Testes Sorológicos , Trypanosoma cruzi , Animais , Cães , Doença de Chagas/diagnóstico , Doença de Chagas/veterinária , Doença de Chagas/parasitologia , Trypanosoma cruzi/imunologia , Trypanosoma cruzi/genética , Doenças do Cão/diagnóstico , Doenças do Cão/parasitologia , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/genética , Testes Sorológicos/métodos , Testes Sorológicos/veterinária , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/veterinária , Anticorpos Antiprotozoários/sangue , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/genéticaRESUMO
In the Americas, P. vivax is the predominant causative species of malaria, a debilitating and economically significant disease. Due to the complexity of the malaria parasite life cycle, a vaccine formulation with multiple antigens expressed in various parasite stages may represent an effective approach. Based on this, we previously designed and constructed a chimeric recombinant protein, PvRMC-1, composed by PvCyRPA, PvCelTOS, and Pvs25 epitopes. This chimeric protein was strongly recognized by naturally acquired antibodies from exposed population in the Brazilian Amazon. However, there was no investigation about the induced immune response of PvRMC-1. Therefore, in this work, we evaluated the immunogenicity of this chimeric antigen formulated in three distinct adjuvants: Stimune, AddaVax or Aluminum hydroxide (Al(OH)3) in BALB/c mice. Our results suggested that the chimeric protein PvRMC-1 were capable to generate humoral and cellular responses across all three formulations. Antibodies recognized full-length PvRMC-1 and linear B-cell epitopes from PvCyRPA, PvCelTOS, and Pvs25 individually. Moreover, mice's splenocytes were activated, producing IFN-γ in response to PvCelTOS and PvCyRPA peptide epitopes, affirming T-cell epitopes in the antigen. While aluminum hydroxide showed notable cellular response, Stimune and Addavax induced a more comprehensive immune response, encompassing both cellular and humoral components. Thus, our findings indicate that PvRMC-1 would be a promising multistage vaccine candidate that could advance to further preclinical studies.
Assuntos
Anticorpos Antiprotozoários , Antígenos de Protozoários , Vacinas Antimaláricas , Malária Vivax , Camundongos Endogâmicos BALB C , Plasmodium vivax , Proteínas de Protozoários , Animais , Plasmodium vivax/imunologia , Plasmodium vivax/genética , Camundongos , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/genética , Malária Vivax/imunologia , Malária Vivax/prevenção & controle , Anticorpos Antiprotozoários/imunologia , Vacinas Antimaláricas/imunologia , Feminino , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/genética , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/genética , Modelos Animais de Doenças , Adjuvantes Imunológicos , Imunogenicidade da Vacina , Antígenos de SuperfícieRESUMO
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/parasitologiaRESUMO
Neosporosis is the major infectious cause of abortion and reproductive losses in cattle worldwide; however, there are no available vaccines or drugs to control this disease. Recently, a dual (positive and negative) DIVA-like (Differentiation of Infected from Vaccinated Animals) vaccine was evaluated in a pregnant mouse model of neosporosis, showing promising immunogenic and protective results. The current report aimed to study the safety, the dose-dependent immunogenicity and the dual DIVA-like character of a recombinant subunit vaccine composed of the major surface antigen from Neospora caninum (rNcSAG1) and the carrier/adjuvant Heat shock protein 81.2 from Arabidopsis thaliana (rAtHsp81.2) in cattle. Healthy heifers were separated and assigned to experimental groups A-F and subcutaneously immunized with 2 doses of vaccine formulations 30 days apart as follows: A (n = 4): 50 µg rNcSAG1 + 150 µg rAtHsp81.2; B (n = 4): 200 µg rNcSAG1 + 600 µg rAtHsp81.2; C (n = 4): 500 µg rNcSAG1 + 1,500 µg rAtHsp81.2; D (n = 3): 150 µg rAtHsp81.2; E (n = 3):1,500 µg rAtHsp81.2, and F (n = 3) 2 ml of sterile PBS. The immunization of heifers with the different vaccine or adjuvant doses (groups A-E) was demonstrated to be safe and did not modify the mean value of the evaluated serum biomarkers of metabolic function (GOT/ASP, GPT/ALT, UREA, Glucose and total proteins). The kinetics and magnitude of the immune responses were dose-dependent. The higher dose of the vaccine formulation (group C) stimulated a broad and potent humoral and cellular immune response, characterized by an IgG1/IgG2 isotype profile and IFN-γ secretion. In addition, this was the first time that dual DIVA-like character of a vaccine against neosporosis was demonstrated, allowing us to differentiate vaccinated from infected heifers by two different DIVA compliant test approaches. These results encourage us to evaluate its protective efficacy in infected pregnant cattle in the future.
Assuntos
Doenças dos Bovinos , Coccidiose , Neospora , Vacinas Protozoárias , Vacinas Sintéticas , Animais , Bovinos , Coccidiose/prevenção & controle , Coccidiose/veterinária , Coccidiose/imunologia , Doenças dos Bovinos/prevenção & controle , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/parasitologia , Neospora/imunologia , Feminino , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Protozoárias/imunologia , Vacinas Protozoárias/administração & dosagem , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Proteínas de Protozoários/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Imunogenicidade da Vacina , GravidezRESUMO
American tegumentary leishmaniasis (ATL) diagnosis is an open question, and the search for a solution is urgent. The available tests that detect the etiological agent of the infection are specific for ATL diagnosis. However, they present disadvantages, such as low sensitivity and the need for invasive procedures to obtain the samples. Immunological methods (leishmanin skin test and search for anti-Leishmania antibodies) are good alternatives to the etiological diagnosis of ATL. Presently, we face problems with disease confirmation due to the discontinuity in the production of leishmanin skin test antigen, particularly in resource-poor settings. Aiming to diagnose ATL, we validated rLb6H-ELISA for IgG antibodies using 1,091 samples from leishmaniasis patients and healthy controls, divided into four panels, living in 19 Brazilian endemic and non-endemic states. The rLb6H-ELISA showed a sensitivity of 98.6% and a specificity of 100.0%, with the reference panel comprising 70 ATL patient samples and 70 healthy controls. The reproducibility evaluation showed a coefficient of variation of positive samples ≤ 8.20% for repeatability, ≤ 17,97% for reproducibility, and ≤ 8.12% for homogeneity. The plates sensitized with rLb6H were stable at 4°C and -20°C for 180 days and 37°C for seven days, indicating 12 months of validity. In samples of ATL patients from five research and healthcare centers in endemic and non-endemic areas, rLb6H-ELISA showed a sensitivity of 84.0%; no significant statistical difference was observed among the five centers (chi-square test, p = 0.13). In samples of healthy controls from four areas with different endemicity, a specificity of 92.4% was obtained; lower specificity was obtained in a visceral leishmaniasis high endemicity locality (chi-square test, p<0.001). Cross-reactivity was assessed in 166 other disease samples with a positivity of 13.9%. Based on the good diagnostic performance and the reproducibility and stability of the antigen, we suggest using ELISA-rLb6H to diagnose ATL.
Assuntos
Antígenos de Protozoários , Ensaio de Imunoadsorção Enzimática , Leishmaniose Cutânea , Humanos , Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/epidemiologia , Ensaio de Imunoadsorção Enzimática/métodos , Antígenos de Protozoários/imunologia , Feminino , Masculino , Adulto , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Adolescente , Reprodutibilidade dos Testes , Proteínas Recombinantes/imunologia , Adulto Jovem , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Idoso , Criança , Estudos de Casos e Controles , Brasil/epidemiologiaRESUMO
BACKGROUND: Plasmodium falciparum malaria is a public health issue mostly seen in tropical countries. Until now, there is no effective malaria vaccine against antigens specific to the blood-stage of P. falciparum infection. Because the pathogenesis of malarial disease results from blood-stage infection, it is essential to identify the most promising blood-stage vaccine candidate antigens under natural exposure to malaria infection. METHODS: A cohort of 400 pregnant women and their infants was implemented in South Benin. An active and passive protocol of malaria surveillance was established during pregnancy and infancy to precisely ascertain malaria infections during the follow-up. Twenty-eight antibody (Ab) responses specific to seven malaria candidate vaccine antigens were repeatedly quantified during pregnancy (3 time points) and infancy (6 time points) in order to study the Ab kinetics and their protective role. Abs were quantified by ELISA and logistic, linear and cox-proportional hazard model were performed to analyse the associations between Ab responses and protection against malaria in mothers and infants, taking into account socio-economic factors and for infants an environmental risk of exposure. RESULTS: The levels of IgM against MSP1, MSP2 and MSP3 showed an early protective response against the onset of symptomatic malaria infections starting from the 18th month of life, whereas no association was found for IgG responses during infancy. In women, some IgG responses tend to be associated with a protection against malaria risk along pregnancy and at delivery, among them IgG3 against GLURP-R0 and IgG2 against MSP1. CONCLUSION: The main finding suggests that IgM should be considered in vaccine designs during infanthood. Investigation of the functional role played by IgM in malaria protection needs further attention.
Assuntos
Anticorpos Antiprotozoários , Antígenos de Protozoários , Imunoglobulina G , Imunoglobulina M , Malária Falciparum , Plasmodium falciparum , Humanos , Feminino , Plasmodium falciparum/imunologia , Malária Falciparum/prevenção & controle , Malária Falciparum/imunologia , Gravidez , Lactente , Imunoglobulina M/sangue , Imunoglobulina G/sangue , Anticorpos Antiprotozoários/sangue , Benin , Antígenos de Protozoários/imunologia , Adulto , Adulto Jovem , Ensaio de Imunoadsorção Enzimática , Recém-Nascido , Complicações Parasitárias na Gravidez/prevenção & controle , Complicações Parasitárias na Gravidez/imunologia , Estudos de CoortesRESUMO
BACKGROUND: Plasmodium vivax represents the most geographically widespread human malaria parasite affecting civilian and military populations in endemic areas. Targeting the pre-erythrocytic (PE) stage of the parasite life cycle is especially appealing for developing P. vivax vaccines as it would prevent disease and transmission. Here, naturally acquired immunity to a panel of P. vivax PE antigens was explored, which may facilitate vaccine development and lead to a better understanding of naturally acquired PE immunity. METHODS: Twelve P. vivax PE antigens orthologous to a panel of P. falciparum antigens previously identified as highly immunogenic in protected subjects after immunization with radiation attenuated sporozoites (RAS) were used for evaluation of humoral and cellular immunity by ELISA and IFN-γ ELISpot. Samples from P. vivax infected individuals (n = 76) from a low endemic malaria region in the Peruvian Amazon Basin were used. RESULTS: In those clinical samples, all PE antigens evaluated showed positive IgG antibody reactivity with a variable prevalence of 58-99% in recently P. vivax diagnosed patients. The magnitude of the IgG antibody response against PE antigens was lower compared with blood stage antigens MSP1 and DBP-II, although antibody levels persisted better for PE antigens (average decrease of 6% for PE antigens and 43% for MSP1, p < 0.05). Higher IgG antibodies was associated with one or more previous malaria episodes only for blood stage antigens (p < 0.001). High IgG responders across PE and blood stage antigens showed significantly lower parasitaemia compared to low IgG responders (median 1,921 vs 4,663 par/µl, p < 0.05). In a subgroup of volunteers (n = 17),positive IFN-γ T cell response by ELISPOT was observed in 35% vs 9-35% against blood stage MSP1 and PE antigens, respectively, but no correlation with IgG responses. CONCLUSIONS: These results demonstrate clear humoral and T cell responses against P. vivax PE antigens in individuals naturally infected with P. vivax. These data identify novel attractive PE antigens suitable for use in the potential development and selection of new malaria vaccine candidates which can be used as a part of malaria prevention strategies in civilian and military populations living in P. vivax endemic areas.
Assuntos
Antígenos de Protozoários , Malária Vivax , Plasmodium vivax , Proteínas de Protozoários , Plasmodium vivax/imunologia , Peru/epidemiologia , Humanos , Malária Vivax/imunologia , Malária Vivax/epidemiologia , Adulto , Masculino , Adulto Jovem , Adolescente , Feminino , Pessoa de Meia-Idade , Proteínas de Protozoários/imunologia , Antígenos de Protozoários/imunologia , Imunoglobulina G/sangue , Anticorpos Antiprotozoários/sangue , Ensaio de Imunoadsorção Enzimática , Criança , Idoso , ELISPOTRESUMO
Serodiagnosis methods have been used as platforms for diagnostic tests for many diseases. Due to magnetic nanoparticles' properties to quickly detach from an external magnetic field and particle size effects, these nanomaterials' functionalization allows the specific isolation of target analytes, enhancing accuracy parameters and reducing serodiagnosis time. Superparamagnetic iron oxide nanoparticles (MNPs) were synthesized and functionalized with polyethylene glycol (PEG) and then associated with the synthetic Leishmaniosis epitope. This nano-peptide antigen showed promising results. Regarding Tegumentary leishmaniasis diagnostic accuracy, the AUC was 0.8398 with sensibility 75% (95CI% 50.50 - 89.82) and specificity 87.50% (95CI% 71.93 - 95.03), and Visceral leishmaniasis accuracy study also present high performance, the AUC was 0.9258 with sensibility 87.50% (95CI% 63.98 - 97.78) and specificity 87.50% (95CI% 71.93 - 95.03). Our results demonstrate that the association of the antigen with MNPs accelerates and improves the diagnosis process. MNPs could be an important tool for enhancing serodiagnosis.