RESUMEN
The clinical manifestations of cutaneous leishmaniasis (CL) depend not only on the infecting species involved, but also on the immune response of the individual. Although not yet well understood in humans, parasite survival and persistence are related to the cytokine profile and T cell proliferation, with the Th1 profile being related to cure, and the Th2 profile to disease progression. Considering the need for studies focused on the species with the highest circulation in the state of Amazonas, this study aimed to analyze the immunoregulation stimulated by soluble antigens (SLAs) of Leishmania (L.) amazonensis and Leishmania (V.) guyanensis in human lymphocytes in vitro, in order to understand the immune response of patients with CL. Lymphoproliferation was evaluated against stimuli of SLAs from L. amazonensis (100 µg/mL), SLAs from L. guyanensis (100 µg/mL) and phytohemagglutinin (10 µg/mL) using a BrdU Cell Proliferation ELISA kit after 72 h of incubation. Quantification of the cytokines IL-1b, IL-6, IL-8, IL-10, IL-12 and TNF was performed using the BD™ cytometric bead array human Th1/Th2/Th17 cytokine kit. Our results demonstrated that soluble antigens from L. amazonensis and L. guyanensis stimulated the lymphoproliferation of PBMCs from patients primo-infected with CL. Among the cytokines dosed, the highest concentrations were of IL-6 and IL-8, thus demonstrating that the soluble antigens evaluated are capable of inducing regulatory mechanisms.
Asunto(s)
Antígenos de Protozoos , Proliferación Celular , Citocinas , Humanos , Antígenos de Protozoos/inmunología , Citocinas/inmunología , Proliferación Celular/efectos de los fármacos , Leishmaniasis Cutánea/inmunología , Adulto , Linfocitos/inmunología , Ensayo de Inmunoadsorción Enzimática , Masculino , Femenino , Leishmania guyanensis/inmunología , Adulto JovenRESUMEN
We developed a protein to rapidly and accurately diagnose Chagas disease, a life-threatening illness identified by the WHO as a critical worldwide public health risk. Limitations in present day serological tests are complicating the current health situation and contributing to most infected persons being unaware of their condition and therefore untreated. To improve diagnostic testing, we developed an immunological mimic of the etiological agent, Trypanosoma cruzi, by combining ten pathogen-specific epitopes within the beta-barrel protein structure of Thermal Green Protein. The resulting multi-epitope protein, DxCruziV3, displayed high specificity and sensitivity as the antibody capture reagent in an ELISA platform with an analytical sensitivity that exceeds WHO recommendations. Within an immunochromatographic platform, DxCruziV3 showed excellent performance for the point of application diagnosis in a region endemic for multiple diseases, the municipality of Barcelos in the state of Amazonas, Brazil. In total, 167 individuals were rapidly tested using whole blood from a finger stick. As recommended by the Brazilian Ministry of Health, venous blood samples were laboratory tested by conventional assays for comparison. Test results suggest utilizing DxCruziV3 in different assay platforms can confidently diagnose chronic infections by T. cruzi. Rapid and more accurate results will benefit everyone but will have the most noticeable impact in resource-limited rural areas where the disease is endemic.
Asunto(s)
Enfermedad de Chagas , Ensayo de Inmunoadsorción Enzimática , Epítopos , Pruebas Serológicas , Trypanosoma cruzi , Enfermedad de Chagas/diagnóstico , Enfermedad de Chagas/sangre , Enfermedad de Chagas/inmunología , Humanos , Ensayo de Inmunoadsorción Enzimática/métodos , Trypanosoma cruzi/inmunología , Pruebas Serológicas/métodos , Epítopos/inmunología , Enfermedad Crónica , Masculino , Sensibilidad y Especificidad , Femenino , Adulto , Anticuerpos Antiprotozoarios/sangre , Anticuerpos Antiprotozoarios/inmunología , Persona de Mediana Edad , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/sangre , Brasil/epidemiologíaRESUMEN
BACKGROUND: Laboratory diagnosis of American cutaneous leishmaniasis (ACL) requires a tool amenable to the epidemiological status of ACL in Brazil. Montenegro skin test (MST), an efficient immunological tool used for laboratory diagnosis of ACL, induces delayed-type hypersensitivity (DTH) response to the promastigote antigens of Leishmania; however, human immune responses against infection are modulated by the amastigote of the parasite. Leishmania (V.) lainsoni induces strong cellular immunity in humans; therefore, the antigenic reactivity of its axenic amastigote (AMA antigen) to MST was evaluated for the laboratory diagnosis of ACL. METHODS: Among 70 individuals examined, 60 had a laboratory-confirmed diagnosis of ACL; 53 had localized cutaneous leishmaniasis (LCL), and 7 had mucosal leishmaniasis (ML). Patients were treated at the Evandro Chagas Institute's leishmaniasis clinic, Pará State, Brazil. Ten healthy individuals with no history of ACL (control group) were also examined. Leishmania (V.) braziliensis promastigote antigen (PRO) was used to compare the reactivity with that of AMA antigen. Paired Student's t-test, kappa agreement, and Spearman test were used to evaluate the reactivity of AMA and PRO. RESULTS: The mean reactivity of AMA in ACL patients was 19.4 mm ± 13.3, which was higher (P < 0.001) than that of PRO: 12.1 mm ± 8.1. MST reactivity according to the clinical forms revealed that AMA reactivity in LCL and ML, 18.8 mm ± 13.3 and 24.3 mm ± 13.7, was higher (P < 0.001) than that of PRO, 11.8 mm ± 8.2 and 14.6 mm ± 8.4, respectively. CONCLUSION: AMA reactivity was higher than that of PRO, indicating that AMA is a promising alternative for optimizing MST in the laboratory diagnosis of ACL.
Asunto(s)
Antígenos de Protozoos , Leishmania , Leishmaniasis Cutánea , Pruebas Cutáneas , Humanos , Antígenos de Protozoos/inmunología , Pruebas Cutáneas/métodos , Adulto , Femenino , Leishmaniasis Cutánea/diagnóstico , Leishmaniasis Cutánea/inmunología , Leishmaniasis Cutánea/parasitología , Masculino , Brasil , Leishmania/inmunología , Persona de Mediana Edad , Adulto Joven , AdolescenteRESUMEN
Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a surface protein found in two stages of the malaria life cycle. This is a protein involved in a reorientation movement of the parasite so that cell invasion occurs in the so-called "moving junction", relevant when the membranes of the parasite and the host are in contact. The structure of a conformational epitope of domain III of PfAMA1 in complex with the monoclonal antibody Fab F8.12.19 is experimentally known. Here, we used molecular dynamics with enhanced sampling by Hamiltonian replica exchange molecular dynamics (HREMD) to understand the effect of intermolecular interactions, conformational variability, and intrinsically disordered regions on the mechanism of antigen-antibody interaction. Clustering methods and the analysis of conformational variability were used in order to understand the influence of the presence of the partner protein in the complex. The free-state epitope accesses a broader conformational pool, including disordered conformations not seen in the bound state. The simulations suggest an extended conformational selection mechanism in which the antibody stabilizes a conformational set of the epitope existing in the free state. The stabilization of the active conformation occurs mainly through hydrogen bonds: Tyr(H33)-Asp493, His(L94)-Val510, Ser(L93)-Glu511, Tyr(H56)-Asp485, and Tyr(H35)-Asp493. The antibody has a structure with few flexible regions, and only the complementarity determining region (CDR) H3 shows greater plasticity in the presence of the epitope.
Asunto(s)
Antígenos de Protozoos , Proteínas de la Membrana , Simulación de Dinámica Molecular , Plasmodium falciparum , Proteínas Protozoarias , Proteínas Protozoarias/química , Proteínas Protozoarias/inmunología , Antígenos de Protozoos/química , Antígenos de Protozoos/inmunología , Proteínas de la Membrana/química , Proteínas de la Membrana/inmunología , Plasmodium falciparum/inmunología , Plasmodium falciparum/química , Conformación Proteica , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunologíaRESUMEN
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.
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Adyuvantes Inmunológicos , Linfocitos T CD4-Positivos , Linfocitos T CD8-positivos , Epítopos de Linfocito T , Memoria Inmunológica , Vacunas contra la Leishmaniasis , Leishmaniasis Cutánea , Animales , Leishmaniasis Cutánea/prevención & control , Leishmaniasis Cutánea/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD4-Positivos/inmunología , Epítopos de Linfocito T/inmunología , Ratones , Vacunas contra la Leishmaniasis/inmunología , Femenino , Adyuvantes Inmunológicos/administración & dosificación , Ratones Endogámicos BALB C , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/genética , Leishmania braziliensis/inmunología , Lípido A/análogos & derivados , Lípido A/inmunología , Anticuerpos Antiprotozoarios/inmunología , Citocinas/metabolismo , Citocinas/inmunología , Modelos Animales de Enfermedad , Antígenos de Protozoos/inmunologíaRESUMEN
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.
Asunto(s)
Anticuerpos Antiprotozoarios , Antígenos de Protozoos , Ensayo de Inmunoadsorción Enzimática , Leishmania , Leishmaniasis Cutánea , Proteínas Protozoarias , Proteínas Recombinantes , Sensibilidad y Especificidad , Humanos , Ensayo de Inmunoadsorción Enzimática/métodos , Leishmaniasis Cutánea/diagnóstico , Leishmaniasis Cutánea/orina , Proteínas Protozoarias/orina , Proteínas Protozoarias/inmunología , Antígenos de Protozoos/orina , Antígenos de Protozoos/inmunología , Leishmania/inmunología , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/orina , Adulto , Femenino , Anticuerpos Antiprotozoarios/sangre , Anticuerpos Antiprotozoarios/orina , Masculino , Persona de Mediana Edad , Adulto Joven , Adolescente , Anciano , Orina/química , Orina/parasitología , Niño , Preescolar , Epítopos de Linfocito B/inmunologíaRESUMEN
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.
Asunto(s)
Anticuerpos Antiprotozoarios , Antígenos de Protozoos , Enfermedad de Chagas , Enfermedades de los Perros , Ensayo de Inmunoadsorción Enzimática , Sensibilidad y Especificidad , Pruebas Serológicas , Trypanosoma cruzi , Animales , Perros , Enfermedad de Chagas/diagnóstico , Enfermedad de Chagas/veterinaria , Enfermedad de Chagas/parasitología , Trypanosoma cruzi/inmunología , Trypanosoma cruzi/genética , Enfermedades de los Perros/diagnóstico , Enfermedades de los Perros/parasitología , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/genética , Pruebas Serológicas/métodos , Pruebas Serológicas/veterinaria , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Anticuerpos Antiprotozoarios/sangre , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/genéticaRESUMEN
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.
Asunto(s)
Anticuerpos Antiprotozoarios , Antígenos de Protozoos , Vacunas contra la Malaria , Malaria Vivax , Ratones Endogámicos BALB C , Plasmodium vivax , Proteínas Protozoarias , Animales , Plasmodium vivax/inmunología , Plasmodium vivax/genética , Ratones , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/genética , Malaria Vivax/inmunología , Malaria Vivax/prevención & control , Anticuerpos Antiprotozoarios/inmunología , Vacunas contra la Malaria/inmunología , Femenino , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/genética , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito B/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/genética , Modelos Animales de Enfermedad , Adyuvantes Inmunológicos , Inmunogenicidad Vacunal , Antígenos de SuperficieRESUMEN
Hard-to-reach communities represent Peru's main challenge for malaria elimination, but information about transmission in these areas is scarce. Here, we assessed Plasmodium vivax (Pv) and P. falciparum (Pf) transmission dynamics, resistance markers, and Pf hrp2/3 deletions in Nueva Jerusalén (NJ), a remote, indigenous community in the Peruvian Amazon with high population mobility. We collected samples from November 2019 to May 2020 by active (ACD) and passive case detection (PCD) in NJ. Parasites were identified with microscopy and PCR. Then, we analyzed a representative set of positive-PCR samples (Pv = 68, Pf = 58) using highly-multiplexed deep sequencing assays (AmpliSeq) and compared NJ parasites with ones from other remote Peruvian areas using population genetics indexes. The ACD intervention did not reduce malaria cases in the short term, and persistent malaria transmission was observed (at least one Pv infection was detected in 96% of the study days). In Nueva Jerusalen, the Pv population had modest genetic diversity (He = 0.27). Pf population had lower diversity (He = 0.08) and presented temporal clustering, one of these clusters linked to an outbreak in February 2020. Moreover, Pv and Pf parasites from NJ exhibited variable levels of differentiation (Pv Fst = 0.07-0.52 and Pf Fst = 0.11-0.58) with parasites from other remote areas. No artemisin resistance mutations but chloroquine (57%) and sulfadoxine-pyrimethamine (35-67%) were detected in NJ's Pf parasites. Moreover, pfhrp2/3 gene deletions were common (32-50% of parasites with one or both genes deleted). The persistent Pv transmission and the detection of a Pf outbreak with parasites genetically distinct from the local ones highlight the need for tailored interventions focusing on mobility patterns and imported infections in remote areas to eliminate malaria in the Peruvian Amazon.
Asunto(s)
Malaria Falciparum , Malaria Vivax , Plasmodium falciparum , Plasmodium vivax , Proteínas Protozoarias , Perú/epidemiología , Humanos , Plasmodium falciparum/genética , Plasmodium falciparum/aislamiento & purificación , Plasmodium vivax/genética , Plasmodium vivax/aislamiento & purificación , Malaria Falciparum/epidemiología , Malaria Falciparum/parasitología , Malaria Falciparum/transmisión , Malaria Vivax/epidemiología , Malaria Vivax/parasitología , Malaria Vivax/transmisión , Proteínas Protozoarias/genética , Femenino , Masculino , Niño , Adulto , Antimaláricos/uso terapéutico , Antimaláricos/farmacología , Adolescente , Resistencia a Medicamentos/genética , Persona de Mediana Edad , Pueblos Indígenas/genética , Adulto Joven , Preescolar , Genómica/métodos , Variación Genética , Antígenos de Protozoos/genéticaRESUMEN
In malaria parasites, the erythrocyte binding-like proteins (EBL) are a family of invasion proteins that are attractive vaccine targets. In the case of Plasmodium vivax, the widespread malaria parasite, blood-stage vaccines have been largely focused on a single EBL candidate, the Duffy binding-like domain (DBL) of the Duffy binding protein (DBPII), due to its well-characterized role in the reticulocyte invasion. A novel P. vivax EBL family member, the Erythrocyte binding protein (EBP2, also named EBP or DBP2), binds preferentially to reticulocytes and may mediate an alternative P. vivax invasion pathway. To gain insight into the natural genetic diversity of the DBL domain of EBP2 (region II; EBP2-II), we analyzed ebp2-II gene sequences of 71 P. vivax isolates collected in different endemic settings of the Brazilian Amazon rainforest, where P. vivax is the predominant malaria-associated species. Although most of the substitutions in the ebp2-II gene were non-synonymous and suggested positive selection, the results showed that the DBL domain of the EBP2 was much less polymorphic than that of DBPII. The predominant EBP2 haplotype in the Amazon region corresponded to the C127 reference sequence first described in Cambodia (25% C127-like haplotype). An overview of ebp2-II gene sequences available at GenBank (n = 352) from seven countries (Cambodia, Madagascar, Myanmar, PNG, South Korea, Thailand, Vietnam) confirmed the C127-like haplotype as highly prevalent worldwide. Two out of 43 haplotypes (5 to 20 inferred per country) showed a global frequency of 60%. The results presented here open new avenues of research pursuit while suggesting that a vaccine based on the DBL domain of EBP2 should target a few haplotypes for broad coverage.
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Variación Genética , Malaria Vivax , Plasmodium vivax , Proteínas Protozoarias , Plasmodium vivax/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/química , Malaria Vivax/parasitología , Humanos , Bosque Lluvioso , Filogenia , Haplotipos , Antígenos de Protozoos/genética , Dominios Proteicos , Receptores de Superficie CelularRESUMEN
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.
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Antígenos de Protozoos , Ensayo de Inmunoadsorción Enzimática , Leishmaniasis Cutánea , Humanos , Leishmaniasis Cutánea/diagnóstico , Leishmaniasis Cutánea/inmunología , Leishmaniasis Cutánea/parasitología , Leishmaniasis Cutánea/epidemiología , Ensayo de Inmunoadsorción Enzimática/métodos , Antígenos de Protozoos/inmunología , Femenino , Masculino , Adulto , Persona de Mediana Edad , Sensibilidad y Especificidad , Adolescente , Reproducibilidad de los Resultados , Proteínas Recombinantes/inmunología , Adulto Joven , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Anticuerpos Antiprotozoarios/sangre , Anticuerpos Antiprotozoarios/inmunología , Anciano , Niño , Estudios de Casos y Controles , Brasil/epidemiologíaRESUMEN
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.
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Antígenos de Protozoos , Enfermedades de los Bovinos , Ensayo de Inmunoadsorción Enzimática , Pruebas Serológicas , Trypanosoma vivax , Animales , Bovinos , Argentina/epidemiología , Trypanosoma vivax/inmunología , Trypanosoma vivax/genética , Trypanosoma vivax/aislamiento & purificación , Pruebas Serológicas/métodos , Pruebas Serológicas/veterinaria , Enfermedades de los Bovinos/diagnóstico , Enfermedades de los Bovinos/epidemiología , Enfermedades de los Bovinos/parasitología , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/genética , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Anticuerpos Antiprotozoarios/sangre , Sensibilidad y Especificidad , Tripanosomiasis Africana/diagnóstico , Tripanosomiasis Africana/veterinaria , Tripanosomiasis Africana/epidemiología , Ganado/parasitologíaRESUMEN
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.
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Enfermedades de los Bovinos , Coccidiosis , Neospora , Vacunas Antiprotozoos , Vacunas Sintéticas , Animales , Bovinos , Coccidiosis/prevención & control , Coccidiosis/veterinaria , Coccidiosis/inmunología , Enfermedades de los Bovinos/prevención & control , Enfermedades de los Bovinos/inmunología , Enfermedades de los Bovinos/parasitología , Neospora/inmunología , Femenino , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Antiprotozoos/inmunología , Vacunas Antiprotozoos/administración & dosificación , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/inmunología , Proteínas Protozoarias/inmunología , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/administración & dosificación , Inmunogenicidad Vacunal , EmbarazoRESUMEN
Toxoplasmosis is the most prevalent parasitic zoonosis worldwide, causing ocular and neurological diseases. No vaccine has been approved for human use. We evaluated the response of peripheral blood mononuclear cells (PBMCs) to a novel construct of Toxoplasma gondii total antigen in maltodextrin nanoparticles (NP/TE) in individuals with varying infectious statuses (uninfected, chronic asymptomatic, or ocular toxoplasmosis). We analyzed the concentration of IFN-γ after NP/TE ex vivo stimulation using ELISA and the immunophenotypes of CD4+ and CD8+ cell populations using flow cytometry. In addition, serotyping of individuals with toxoplasmosis was performed by ELISA using GRA6-derived polypeptides. Low doses of NP/TE stimulation (0.9 µg NP/0.3 µg TE) achieved IFN-γ-specific production in previously exposed human PBMCs without significant differences in the infecting serotype. Increased IFN-γ expression in CD4+ effector memory cell subsets was found in patients with ocular toxoplasmosis with NP/TE but not with TE alone. This is the first study to show how T-cell subsets respond to ex vivo stimulation with a vaccine candidate for human toxoplasmosis, providing crucial insights for future clinical trials.
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Antígenos de Protozoos , Interferón gamma , Activación de Linfocitos , Nanopartículas , Polisacáridos , Toxoplasma , Toxoplasmosis , Humanos , Nanopartículas/química , Polisacáridos/inmunología , Toxoplasma/inmunología , Antígenos de Protozoos/inmunología , Toxoplasmosis/inmunología , Interferón gamma/metabolismo , Interferón gamma/inmunología , Activación de Linfocitos/inmunología , Femenino , Adulto , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Masculino , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Persona de Mediana EdadRESUMEN
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.
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Antígenos de Protozoos , Malaria Vivax , Plasmodium vivax , Proteínas Protozoarias , Plasmodium vivax/inmunología , Perú/epidemiología , Humanos , Malaria Vivax/inmunología , Malaria Vivax/epidemiología , Adulto , Masculino , Adulto Joven , Adolescente , Femenino , Persona de Mediana Edad , Proteínas Protozoarias/inmunología , Antígenos de Protozoos/inmunología , Inmunoglobulina G/sangre , Anticuerpos Antiprotozoarios/sangre , Ensayo de Inmunoadsorción Enzimática , Niño , Anciano , Ensayo de Immunospot Ligado a EnzimasRESUMEN
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.
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Anticuerpos Antiprotozoarios , Antígenos de Protozoos , Inmunoglobulina G , Inmunoglobulina M , Malaria Falciparum , Plasmodium falciparum , Humanos , Femenino , Plasmodium falciparum/inmunología , Malaria Falciparum/prevención & control , Malaria Falciparum/inmunología , Embarazo , Lactante , Inmunoglobulina M/sangre , Inmunoglobulina G/sangre , Anticuerpos Antiprotozoarios/sangre , Benin , Antígenos de Protozoos/inmunología , Adulto , Adulto Joven , Ensayo de Inmunoadsorción Enzimática , Recién Nacido , Complicaciones Parasitarias del Embarazo/prevención & control , Complicaciones Parasitarias del Embarazo/inmunología , Estudios de CohortesRESUMEN
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.
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Ensayo de Inmunoadsorción Enzimática , Polietilenglicoles , Sensibilidad y Especificidad , Humanos , Ensayo de Inmunoadsorción Enzimática/métodos , Polietilenglicoles/química , Antígenos de Protozoos/inmunología , Leishmaniasis/diagnóstico , Nanopartículas Magnéticas de Óxido de Hierro/química , Anticuerpos Antiprotozoarios/sangreRESUMEN
Chagas disease, caused by the protozoa Trypanosoma cruzi, continues to be a serious public health problem in Latin America, worsened by the limitations in its detection. Given the importance of developing new diagnostic methods for this disease, the present review aimed to verify the number of publications dedicated to research on peptides that demonstrate their usefulness in serodiagnosis. To this end, a bibliographic survey was conducted on the PubMed platform using the keyword "peptide" or "epitope" combined with "Chagas disease" or "Trypanosoma cruzi"; "diagno*" or "serodiagnosis" or "immunodiagnosis", without period restriction. An increasing number of publications on studies employing peptides in ELISA and rapid tests assays was verified, which confirms the expansion of research in this field. It is possible to observe that many of the peptides tested so far originate from proteins widely used in the diagnosis of Chagas, and many of them are part of commercial tests developed. In this sense, as expected, promising results were obtained for several peptides when tested in ELISA, as many of them exhibited sensitivity and specificity values above 90%. Furthermore, some peptides have been tested in several studies, confirming their diagnostic potential. Despite the promising results observed, it is possible to emphasize the need for extensive testing of peptides, using different serological panels, in order to confirm their potential. The importance of producing an effective assay capable of detecting the clinical stages of the disease, as well as new immunogenic antigens that enable new serological diagnostic tools for Chagas disease, is evident.
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Enfermedad de Chagas , Ensayo de Inmunoadsorción Enzimática , Péptidos , Trypanosoma cruzi , Enfermedad de Chagas/diagnóstico , Enfermedad de Chagas/inmunología , Enfermedad de Chagas/sangre , Humanos , Trypanosoma cruzi/inmunología , Péptidos/inmunología , Péptidos/química , Ensayo de Inmunoadsorción Enzimática/métodos , Pruebas Inmunológicas/métodos , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/sangre , Pruebas Serológicas/métodosRESUMEN
Malaria remains a public health challenge. Since many control strategies have proven ineffective in eradicating this disease, new strategies are required, among which the design of a multivalent vaccine stands out. However, the effectiveness of this strategy has been hindered, among other reasons, by the genetic diversity observed in parasite antigens. In Plasmodium vivax, the Erythrocyte Binding Protein (PvEBP, also known as DBP2) is an alternate ligand to Duffy Binding Protein (DBP); given its structural resemblance to DBP, EBP/DBP2 is proposed as a promising antigen for inclusion in vaccine design. However, the extent of genetic diversity within the locus encoding this protein has not been comprehensively assessed. Thus, this study aimed to characterize the genetic diversity of the locus encoding the P. vivax EBP/DBP2 protein and to determine the evolutionary mechanisms modulating this diversity. Several intrapopulation genetic variation parameters were estimated using 36 gene sequences of PvEBP/DBP2 from Colombian P. vivax clinical isolates and 186 sequences available in databases. The study then evaluated the worldwide genetic structure and the evolutionary forces that may influence the observed patterns of genetic variation. It was found that the PvEBP/DBP2 gene exhibits one of the lowest levels of genetic diversity compared to other vaccine-candidate antigens. Four major haplotypes were shared worldwide. Analysis of the protein's 3D structure and epitope prediction identified five regions with potential antigenic properties. The results suggest that the PvEBP/DBP2 protein possesses ideal characteristics to be considered when designing a multivalent effective antimalarial vaccine against P. vivax.
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Antígenos de Protozoos , Variación Genética , Vacunas contra la Malaria , Malaria Vivax , Plasmodium vivax , Proteínas Protozoarias , Plasmodium vivax/genética , Plasmodium vivax/inmunología , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Humanos , Vacunas contra la Malaria/inmunología , Vacunas contra la Malaria/genética , Malaria Vivax/prevención & control , Malaria Vivax/parasitología , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Colombia , Filogenia , Receptores de Superficie CelularRESUMEN
OBJECTIVES: The accuracy of malaria rapid diagnostic tests is threatened by Plasmodium falciparum with pfhrp2/3 deletions. This study compares gene deletion prevalence determined by multiplex real time polymerase chain reaction (qPCR) and conventional polymerase chain reaction (cPCR) using existing samples with clonality previously determined by microsatellite genotyping. METHODS: Multiplex qPCR was used to estimate prevalence of pfhrp2/3 deletions in three sets of previously collected patient samples from Eritrea and Peru. The qPCR was validated by multiplex digital polymerase chain reaction. Sample classification was compared with cPCR, and receiver operating characteristic curve analysis was used to determine the optimal ΔCq threshold that aligned the results of the two assays. RESULTS: qPCR classified 75% (637 of 849) of samples as single, and 212 as mixed-pfhrp2/3 genotypes, with a positive association between clonality and proportion of mixed-pfhrp2/3 genotype samples. The sample classification agreement between cPCR and qPCR was 75.1% (95% confidence interval [CI] 68.6-80.7%) and 47.8% (95% CI 38.9-56.9%) for monoclonal and polyclonal infections. The qPCR prevalence estimates of pfhrp2/3 deletions showed almost perfect (κ = 0.804, 95% CI 0.714-0.895) and substantial agreement (κ = 0.717, 95% CI 0.562-0.872) with cPCR for Peru and 2016 Eritrean samples, respectively. For 2019 Eritrean samples, the prevalence of double pfhrp2/3 deletions was approximately two-fold higher using qPCR. The optimal threshold for matching the assay results was ΔCq = 3. CONCLUSIONS: Multiplex qPCR and cPCR produce comparable estimates of gene deletion prevalence when monoclonal infections dominate; however, qPCR provides higher estimates where multi-clonal infections are common.