RESUMEN
Malaria sterile immunity has been reproducibly induced by immunization with Plasmodium radiation-attenuated sporozoites (RAS). Analyses of sera from RAS-immunized individuals allowed the identification of P. falciparum antigens, such as the circumsporozoite protein (CSP), the basis for the RTS, S and R21Matrix-M vaccines. Similar advances in P. vivax (Pv) vaccination have been elusive. We previously reported 42% (5/12) of sterile protection in malaria-unexposed, Duffy-positive (Fy +) volunteers immunized with PvRAS followed by a controlled human malaria infection (CHMI). Using a custom protein microarray displaying 515 Pv antigens, we found a significantly higher reactivity to PvCSP and one hypothetical protein (PVX_089630) in volunteers protected against P. vivax infection. In mock-vaccinated Fy + volunteers, a strong antibody response to CHMI was also observed. Although the Fy- volunteers immunized with non-irradiated Pv-infected mosquitoes (live sporozoites) did not develop malaria after CHMI, they recognized a high number of antigens, indicating the temporary presence of asexual parasites in peripheral blood. Together, our findings contribute to the understanding of the antibody response to P. vivax infection and allow the identification of novel parasite antigens as vaccine candidates.Trial registration: ClinicalTrials.gov number: NCT01082341.
Asunto(s)
Vacunas contra la Malaria , Malaria Falciparum , Malaria Vivax , Malaria , Animales , Humanos , Plasmodium vivax , Esporozoítos , Formación de Anticuerpos , Inmunización , Vacunación , Malaria/prevención & control , Malaria Falciparum/parasitología , Malaria Vivax/parasitología , Plasmodium falciparumRESUMEN
BACKGROUND: Leptospirosis is an important zoonotic disease worldwide. Humans usually present a mild non-specific febrile illness, but a proportion of them develop more severe outcomes, such as multi-organ failure, lung hemorrhage and death. Such complications are thought to depend on several factors, including the host immunity. Protective immunity is associated with humoral immune response, but little is known about the immune response mounted during naturally-acquired Leptospira infection. METHODS AND PRINCIPAL FINDINGS: Here, we used protein microarray chip to profile the antibody responses of patients with severe and mild leptospirosis against the complete Leptospira interrogans serovar Copenhageni predicted ORFeome. We discovered a limited number of immunodominant antigens, with 36 antigens specific to patients, of which 11 were potential serodiagnostic antigens, identified at acute phase, and 33 were potential subunit vaccine targets, detected after recovery. Moreover, we found distinct antibody profiles in patients with different clinical outcomes: in the severe group, overall IgM responses do not change and IgG responses increase over time, while both IgM and IgG responses remain stable in the mild patient group. Analyses of individual patients' responses showed that >74% of patients in the severe group had significant IgG increases over time compared to 29% of patients in the mild group. Additionally, 90% of IgM responses did not change over time in the mild group, compared to ~51% in the severe group. CONCLUSIONS: In the present study, we detected antibody profiles associated with disease severity and speculate that patients with mild disease were protected from severe outcomes due to pre-existing antibodies, while patients with severe leptospirosis demonstrated an antibody profile typical of first exposure. Our findings represent a significant advance in the understanding of the humoral immune response to Leptospira infection, and we have identified new targets for the development of subunit vaccines and diagnostic tests.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Leptospira interrogans/inmunología , Leptospirosis/inmunología , Proteoma/análisis , Adolescente , Adulto , Anticuerpos Antibacterianos/inmunología , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina M/sangre , Inmunoglobulina M/inmunología , Leptospira interrogans/genética , Leptospira interrogans/aislamiento & purificación , Leptospira interrogans/fisiología , Leptospirosis/sangre , Leptospirosis/diagnóstico , Leptospirosis/microbiología , Masculino , Análisis por Matrices de Proteínas , Proteoma/inmunología , Pruebas Serológicas , Adulto JovenRESUMEN
BACKGROUND: Acquisition of malaria immunity in low transmission areas usually occurs after relatively few exposures to the parasite. A recent Plasmodium vivax experimental challenge trial in malaria naïve and semi-immune volunteers from Colombia showed that all naïve individuals developed malaria symptoms, whereas semi-immune subjects were asymptomatic or displayed attenuated symptoms. Sera from these individuals were analyzed by protein microarray to identify antibodies associated with clinical protection. METHODOLOGY/PRINCIPAL FINDINGS: Serum samples from naïve (n = 7) and semi-immune (n = 9) volunteers exposed to P. vivax sporozoite-infected mosquito bites were probed against a custom protein microarray displaying 515 P. vivax antigens. The array revealed higher serological responses in semi-immune individuals before the challenge, although malaria naïve individuals also had pre-existing antibodies, which were higher in Colombians than US adults (control group). In both experimental groups the response to the P. vivax challenge peaked at day 45 and returned to near baseline at day 145. Additional analysis indicated that semi-immune volunteers without fever displayed a lower response to the challenge, but recognized new antigens afterwards. CONCLUSION: Clinical protection against experimental challenge in volunteers with previous P. vivax exposure was associated with elevated pre-existing antibodies, an attenuated serological response to the challenge and reactivity to new antigens.
Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Malaria Vivax/sangre , Plasmodium vivax/inmunología , Esporozoítos/inmunología , Antígenos de Protozoos/inmunología , Antimaláricos/uso terapéutico , Cloroquina/uso terapéutico , Humanos , Malaria Vivax/inmunología , Análisis por Matrices de ProteínasRESUMEN
With increasing efficiency, accuracy, and speed we can access complete genome sequences from thousands of infectious microorganisms; however, the ability to predict antigenic targets of the immune system based on amino acid sequence alone is still needed. Here we use a Leptospira interrogans microarray expressing 91% (3359) of all leptospiral predicted ORFs (3667) and make an empirical accounting of all antibody reactive antigens recognized in sera from naturally infected humans; 191 antigens elicited an IgM or IgG response, representing 5% of the whole proteome. We classified the reactive antigens into 26 annotated COGs (clusters of orthologous groups), 26 JCVI Mainrole annotations, and 11 computationally predicted proteomic features. Altogether, 14 significantly enriched categories were identified, which are associated with immune recognition including mass spectrometry evidence of in vitro expression and in vivo mRNA up-regulation. Together, this group of 14 enriched categories accounts for just 25% of the leptospiral proteome but contains 50% of the immunoreactive antigens. These findings are consistent with our previous studies of other Gram-negative bacteria. This genome-wide approach provides an empirical basis to predict and classify antibody reactive antigens based on structural, physical-chemical, and functional proteomic features and a framework for understanding the breadth and specificity of the immune response to L. interrogans.
Asunto(s)
Antígenos Bacterianos/sangre , Leptospira interrogans/inmunología , Leptospira interrogans/metabolismo , Leptospirosis/sangre , Proteoma/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Humanos , Leptospira interrogans/genética , Espectrometría de Masas , Análisis por Micromatrices , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Proteoma/genética , Análisis de Secuencia de ADNRESUMEN
Schistosomiasis is a neglected tropical disease that is responsible for almost 300,000 deaths annually. Mass drug administration (MDA) is used worldwide for the control of schistosomiasis, but chemotherapy fails to prevent reinfection with schistosomes, so MDA alone is not sufficient to eliminate the disease, and a prophylactic vaccine is required. Herein, we take advantage of recent advances in systems biology and longitudinal studies in schistosomiasis endemic areas in Brazil to pilot an immunomics approach to the discovery of schistosomiasis vaccine antigens. We selected mostly surface-derived proteins, produced them using an in vitro rapid translation system and then printed them to generate the first protein microarray for a multi-cellular pathogen. Using well-established Brazilian cohorts of putatively resistant (PR) and chronically infected (CI) individuals stratified by the intensity of their S. mansoni infection, we probed arrays for IgG subclass and IgE responses to these antigens to detect antibody signatures that were reflective of protective vs. non-protective immune responses. Moreover, probing for IgE responses allowed us to identify antigens that might induce potentially deleterious hypersensitivity responses if used as subunit vaccines in endemic populations. Using multi-dimensional cluster analysis we showed that PR individuals mounted a distinct and robust IgG1 response to a small set of newly discovered and well-characterized surface (tegument) antigens in contrast to CI individuals who mounted strong IgE and IgG4 responses to many antigens. Herein, we show the utility of a vaccinomics approach that profiles antibody responses of resistant individuals in a high-throughput multiplex approach for the identification of several potentially protective and safe schistosomiasis vaccine antigens.
Asunto(s)
Anticuerpos Antihelmínticos/sangre , Antígenos Helmínticos/inmunología , Resistencia a la Enfermedad/inmunología , Esquistosomiasis/inmunología , Vacunas/inmunología , Adolescente , Adulto , Anticuerpos Antihelmínticos/inmunología , Brasil/epidemiología , Enfermedad Crónica , Análisis por Conglomerados , Enfermedades Endémicas , Femenino , Ensayos Analíticos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Enfermedades Desatendidas/inmunología , Análisis por Matrices de Proteínas , Esquistosomiasis/sangre , Esquistosomiasis/epidemiología , Adulto JovenRESUMEN
Brucella melitensis, one of the causative agents of human brucellosis, causes acute, chronic, and relapsing infection. While T cell immunity in brucellosis has been extensively studied in mice, no recognized human T cell epitopes that might provide new approaches to classifying and prognosticating B. melitensis infection have ever been delineated. Twenty-seven pools of 500 major histocompatibility complex class II (MHC-II) restricted peptides were created by computational prediction of promiscuous MHC-II CD4(+) T cell derived from the top 50 proteins recognized by IgG in human sera on a genome level B. melitensis protein microarray. Gamma interferon (IFN-γ) and interleukin-5 (IL-5) enzyme-linked immunospot (ELISPOT) analyses were used to quantify and compare Th1 and Th2 responses of leukapheresis-obtained peripheral blood mononuclear cells from Peruvian subjects cured after acute infection (n = 9) and from patients who relapsed (n = 5). Four peptide epitopes derived from 3 B. melitensis proteins (BMEI 1330, a DegP/HtrA protease; BMEII 0029, type IV secretion system component VirB5; and BMEII 0691, a predicted periplasmic binding protein of a peptide transport system) were found repeatedly to produce significant IFN-γ ELISPOT responses in both acute-infection and relapsing patients; none of the peptides distinguished the patient groups. IL-5 responses against the panel of peptides were insignificant. These experiments are the first to systematically identify B. melitensis MHC-II-restricted CD4(+) T cell epitopes recognized by the human immune response, with the potential for new approaches to brucellosis diagnostics and understanding the immunopathogenesis related to this intracellular pathogen.
Asunto(s)
Brucella melitensis/inmunología , Brucelosis/inmunología , Linfocitos T CD4-Positivos/inmunología , Epítopos de Linfocito T/inmunología , Enfermedad Aguda , Antígenos Bacterianos/análisis , Proteínas Bacterianas/análisis , Enfermedad Crónica , Estudios de Cohortes , Humanos , Inmunidad Celular/inmunología , Inmunoglobulina G/inmunología , Interferón gamma/metabolismo , Interleucina-5/metabolismo , Análisis por Micromatrices/métodos , PerúRESUMEN
The development of pre-erythrocytic Plasmodium vivax vaccines is hindered by the lack of in vitro culture systems or experimental rodent models. To help bypass these roadblocks, we exploited the fact that naturally exposed Fy- individuals who lack the Duffy blood antigen (Fy) receptor are less likely to develop blood-stage infections; therefore, they preferentially develop immune responses to pre-erythrocytic-stage parasites, whereas Fy+ individuals experience both liver- and blood-stage infections and develop immune responses to both pre-erythrocytic and erythrocytic parasites. We screened 60 endemic sera from P. vivax-exposed Fy+ or Fy- donors against a protein microarray containing 91 P. vivax proteins with P. falciparum orthologs that were up-regulated in sporozoites. Antibodies against 10 P. vivax antigens were identified in sera from P. vivax-exposed individuals but not unexposed controls. This technology has promising implications in the discovery of potential vaccine candidates against P. vivax malaria.
Asunto(s)
Antígenos de Protozoos/aislamiento & purificación , Sistema del Grupo Sanguíneo Duffy/inmunología , Eritrocitos/parasitología , Malaria Vivax/parasitología , Plasmodium vivax/inmunología , Anticuerpos Antiprotozoarios/sangre , Anticuerpos Antiprotozoarios/inmunología , Formación de Anticuerpos , Antígenos de Protozoos/genética , Colombia , Biología Computacional , Eritrocitos/inmunología , Perfilación de la Expresión Génica , Ensayos Analíticos de Alto Rendimiento , Humanos , Hígado/parasitología , Hígado/patología , Vacunas contra la Malaria/inmunología , Plasmodium vivax/genética , Plasmodium vivax/crecimiento & desarrollo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Esporozoítos/inmunología , Esporozoítos/metabolismo , Regulación hacia ArribaRESUMEN
Brucellosis is a widespread zoonotic disease that is also a potential agent of bioterrorism. Current serological assays to diagnose human brucellosis in clinical settings are based on detection of agglutinating anti-LPS antibodies. To better understand the universe of antibody responses that develop after B. melitensis infection, a protein microarray was fabricated containing 1,406 predicted B. melitensis proteins. The array was probed with sera from experimentally infected goats and naturally infected humans from an endemic region in Peru. The assay identified 18 antigens differentially recognized by infected and non-infected goats, and 13 serodiagnostic antigens that differentiate human patients proven to have acute brucellosis from syndromically similar patients. There were 31 cross-reactive antigens in healthy goats and 20 cross-reactive antigens in healthy humans. Only two of the serodiagnostic antigens and eight of the cross-reactive antigens overlap between humans and goats. Based on these results, a nitrocellulose line blot containing the human serodiagnostic antigens was fabricated and applied in a simple assay that validated the accuracy of the protein microarray results in the diagnosis of humans. These data demonstrate that an experimentally infected natural reservoir host produces a fundamentally different immune response than a naturally infected accidental human host.