APRANK: Computational Prioritization of Antigenic Proteins and Peptides From Complete Pathogen Proteomes.

Availability of highly parallelized immunoassays has renewed interest in the discovery of serology biomarkers for infectious diseases. Protein and peptide microarrays now provide a rapid, high-throughput platform for immunological testing and validation of potential antigens and B-cell epitopes. However, there is still a need for tools to prioritize and select relevant probes when designing these arrays. In this work we describe a computational method called APRANK (Antigenic Protein and Peptide Ranker) which integrates multiple molecular features to prioritize potentially antigenic proteins and peptides in a given pathogen proteome. These features include subcellular localization, presence of repetitive motifs, natively disordered regions, secondary structure, transmembrane spans and predicted interaction with the immune system. We trained and tested this method with a number of bacteria and protozoa causing human diseases: Borrelia burgdorferi (Lyme disease), Brucella melitensis (Brucellosis), Coxiella burnetii (Q fever), Escherichia coli (Gastroenteritis), Francisella tularensis (Tularemia), Leishmania braziliensis (Leishmaniasis), Leptospira interrogans (Leptospirosis), Mycobacterium leprae (Leprae), Mycobacterium tuberculosis (Tuberculosis), Plasmodium falciparum (Malaria), Porphyromonas gingivalis (Periodontal disease), Staphylococcus aureus (Bacteremia), Streptococcus pyogenes (Group A Streptococcal infections), Toxoplasma gondii (Toxoplasmosis) and Trypanosoma cruzi (Chagas Disease). We have evaluated this integrative method using non-parametric ROC-curves and made an unbiased validation using Onchocerca volvulus as an independent data set. We found that APRANK is successful in predicting antigenicity for all pathogen species tested, facilitating the production of antigen-enriched protein subsets. We make APRANK available to facilitate the identification of novel diagnostic antigens in infectious diseases.

Desempenho dos antígenos PGL-I, LID-1e NDO-LID para diagnóstico sorológico de hanseníase em pacientes e contatos domiciliares: revisão de literatura / Performance of PGL-I, LID-1 and NDO-LID antigens for leprosy serological diagnosis in patients and household contacts: a review of the literature

A hanseníase afeta os nervos periféri-cos e a pele levando a ocorrência de incapacidades na ausência de tratamento específico oportuno. Portanto, parâmetros sorológicos são necessários para intervenções terapêuticas precoces. A detecção de anticorpos contra o glicolipídio fenólico I (PGL-I) é amplamente empregada no diagnóstico e classificação clínica, enquanto a proteína Leprosy IDRI Diagnostic (LID)-1 foi desenhada com a intenção de melhorar o diagnóstico de pacien-tes paucibacilares. Posteriormente, este antígeno foi conjugado com o na-tural dissacarídeo ligado ao radical oc-til (ND-O) do PGL-I, originando o NDO--LID, para aumentar sua sensibilidade. Nesta revisão, avaliamos 16 estudos, comparando a performance desses três antígenos (PGL-I, LID-1 e NDO--LID) para diagnóstico da hanseníase e avaliação de contatos domiciliares. Verificamos grande variação quanto às populações envolvidas, tamanho das amostras, classificação clínica dos pacientes e metodologia utilizada, dificultando a comparação. Entre os pacientes multibacilares, a positividade anti-PGL-I variou de 54,0 a 96,0%, en-quanto para LID-1 foi de 47,4 a 94,8% e para NDO-LID apresentou níveis de 60,0 a 98,9%. Nos pacientes paucibacilares, a positividade variou de 6,4 a 52,9% quando PGL-I foi utilizado, 4,0 a 60% contra LID-1 e 16,0 a 63,6% frente ao NDO-LID. Para os contatos domiciliares, as respostas anti-PGL-I, LID-1 e NDO-LID foram 13,2%, 21,7% e 22,9%, respectivamente. O antígeno NDO-LID apresentou maior sensibilidade na maioria dos estudos refletindo seu potencial como ferramenta para o diagnóstico da hanseníase, principalmente em pacientes MB, entretanto, o reconhecimento desse antígeno por contatos domiciliares saudáveis reforça o valor da avaliação clínica para o diagnóstico da hanseníase.(au)

Leprosy affects skin and peripheral nerves bringing several disabilities in absence of specific treatment. So that, effective diagnostic tools are required for early therapeutic interventions. Detection of antibodies against phenolic glycolipid I (PGL-I) is widely employed in the diagnosis and clinical classification while the leprosy IDRI diagnostic (LID-1) protein was designed to improve the diagnosis of paucibacillary patients. More recently, this synthetic antigen was conjugated with the natural octyl disaccharide (ND-O) of PGL-I, originating the NDO-LID in order to increase its sensitivity. Here, we evaluate 16 studies, comparing the performance of these three antigens (PGL-I, LID-1 and NDO-LID) for leprosy diagnosis and evaluation of the household contacts. We verified among the different studies high variation regarding to population involved, sample size, clinical classification of patients and methodology used, making difficult the comparison. Among multibacillary patients, anti-PGL-I positivity ranged from 54.0 to 96.0%, while for LID-1 it was between 47.4 to 94.8% and for NDO-LID presented levels from 60 to 98.9%. In paucibacillary patients, responsiveness ranged from 6.4 to 52.9% when PGL-I was used, 4.0 to 60% against LID-1 and 16.0 to 63.6% if NDO-LID was employed. For household contacts, the responseanti-PGL-I, LID-1 and NDO-LID was13.2%, 21.7% and 22.9%, respectively.NDO-LID antigen showed higher sensitivity in most studies reflecting its potential as tool for leprosy diagnosis, mainly of MB patients, however, the recognition of this antigen by healthy household contact reinforces the value of the clinical evaluation to leprosy diagnosis.(au)

Translational Activities to Enable NTD Vaccines.

There is an urgent need to develop new vaccines for tuberculosis, HIV/AIDS, and malaria, as well as for chronic and debilitating infections known as neglected tropical diseases (NTDs). The term "NTD" emerged at the beginning of the new millennium to describe a set of diseases that are characterized as (1) poverty related, (2) endemic to the tropics and subtropics, (3) lacking public health attention and inadequate industrial investment, (4) having poor research funding and a weak research and development (R&D) pipeline, (5) usually associated with high morbidity but low mortality, and (6) often having no safe and long-lasting treatment available. Many additional challenges to the current control and elimination programs for NTDs exist. These include inconsistent performance of diagnostic tests, regional differences in access to treatment and in treatment outcome, lack of integrated surveillance and vector/intermediate host control, and impact of ecological climatic changes particularly in regions where new cases are increasing in previously nonendemic areas. Moreover, the development of NTD vaccines, including those for schistosomiasis, leishmaniasis, leprosy, hookworm, and Chagas disease are being led by nonprofit product development partnerships (PDPs) working in partnership with academic and industrial partners, contract research organizations, and in some instances vaccine manufacturers in developing countries. In this review, we emphasize global efforts to fuel the development of NTD vaccines, the translational activities needed to effectively move promising vaccine candidates to Phase-I clinical trials and some of the hurdles to ensuring their availability to people in the poorest countries of Africa, Asia, Latin America, and the Caribbean.

Evaluación del antígeno recombinante K39 para el serodiagnóstico de leishmaniasis visceral mediante ensayo inmunoenzimático (ELISA) / Evaluation of recombinant K39 antigen for the serological diagnosis of visceral leishmaniasis with enzyme linked immuno-absorbent assay (ELISA)

La leishmaniasis visceral (LV) es una protozoosis causada por parásitos del complejo Leishmania donovani, caracterizada clínicamente por síndrome febril prolongado, hepatoesplenomegalia, anemia y pérdida progresiva de peso, que puede ser mortal especialmente en niños si no es diagnosticada precozmente. Entre los métodos serológicos más utilizados para el diagnóstico de LV se encuentran el ensayo inmunoenzimático (ELISA). En la actualidad se caracterizan antígenos que aumentan la especificidad de las pruebas serológicas, esto se ha logrado con la utilización de antígenos recombinantes como el K39 (rK39). Este estudio evaluó el antígeno rK39 mediante ELISA, para el serodiagnóstico de leishmaniasis visceral. Se utilizaron 100 sueros caracterizados positivos para LV, que presentaron al menos tres de los siguientes criterios diagnósticos: clínica, epidemiología, serología y demostración directa del parásito (extendido de médula ósea). Los controles incluyen 100 sueros caracterizados negativos para LV y 30 positivos a otras patologías: tuberculosis (5), tripanosomiasis (5), toxoplasmosis (5), lepra (5), leishmaniasis cutánea (5), malaria (2) y HIV/VIH (3). El rK39 obtuvo una sensibilidad de 92 por ciento y una especificidad de 100 por ciento, valor predictivo positivo de 100 por ciento y valor predictivo negativo 92,6 por ciento, así mismo, no se observaron reacciones cruzadas con sueros de otras patologías. Al lograr estandarizar y evaluar el antígeno rK39 empleando ELISA, se concluye que es sensible y específico para el serodiagnóstico de la LV. Al incorporarlo en el protocolo de diagnóstico del Laboratorio de Leishmaniasis del Departamento de Parasitología contribuirá a mejor el diagnóstico de esta parasitosis, beneficiando así a pacientes del Estado, Carabobo y su área de influencia

Salivary anti-PGL IgM and IgA titers and serum antibody IgG titers and avidities in leprosy patients and their correlation with time of infection and antigen exposure.

The present work proposed to correlate serum antibody avidity and salivary antibody titers as parameters for time of infection and antigen exposure in a cohort study evaluating leprosy patients in different periods of treatment. Colorimetric enzyme-immunoassays for salivary antibodies, serum antibody IgG titers and avidities were performed in the samples. Anti-PGL-1 IgA and IgM salivary antibodies were significantly higher in multibacillar (MB-L) patients compared to normal controls (p<0.05), but not when compared to borderline tuberculoid (BT) or to paucibacillar (PB-L) patients (p>0.05). A good correlation was found between salivary anti-PGL-1 IgA and IgM levels in MB-L patients (r=0.41, p<0.01). Two out of 33 tested saliva samples from patients who had completed the drug regimen treatment presented positive salivary antibodies. Among non-treated patients, samples with low, medium or high serum IgG antibody avidity were found in similar frequencies. In patients under treatment, most of the serum samples showed low or medium IgG antibody avidity. The treated MB-L patients showed medium or high antibody avidity, except for two, who showed very low antibody avidity results. We suggest that salivary anti-PGL antibodies and serum IgG avidity could be useful for the indication of recent exposure or re-exposure to bacteria after chemotherapy.

Salivary anti-PGL IgM and IgA titers and serum antibody IgG titers and avidities in leprosy patients and their correlation with time of infection and antigen exposure

The present work proposed to correlate serum antibody avidity and salivary antibody titers as parameters for time of infection and antigen exposure in a co-hort study evaluating leprosy patients in different periods of treatment. Colorimetric enzyme-immunoassays for salivary antibodies, serum antibody IgG titers and avidities were performed in the samples. Anti-PGL-1 IgA and IgM salivary antibodies were significantly higher in multibacillar (MB-L) patients compared to normal controls (p<0.05), but not when compared to borderline tuberculoid (BT) or to paucibacillar (PB-L) patients (p>0.05). A good correlation was found between salivary anti-PGL-1 IgA and IgM levels in MB-L patients (r=0.41, p<0.01). Two out of 33 tested saliva samples from patients who had completed the drug regimen treatment presented positive salivary antibodies. Among non-treated patients, samples with low, medium or high serum IgG antibody avidity were found in similar frequencies. In patients under treatment, most of the serum samples showed low or medium IgG antibody avidity. The treated MB-L patients showed medium or high antibody avidity, except for two, who showed very low antibody avidity results. We suggest that salivary anti-PGL antibodies and serum IgG avidity could be useful for the indication of recent exposure or re-exposure to bacteria after chemotherapy.

Indirect immunofluorescence to demonstrate lichen planus specific antigen (LPSA) in lichen planus.

BACKGROUND: Current evidence suggests that lichen planus is an immunological disease. Cytotoxic CD8+ cells in the lesional epidermis recognize a unique antigen called lichen planus specific antigen. This antigen could be demonstrated by indirect immunofluorescence using the patient's serum and autologous lesional skin. AIM: To study indirect immunofluorescence pattern in lichen planus, among Indian patients. METHODS: Twenty-five consecutive patients with the clinical diagnosis of lichen planus were enrolled in the study. Direct immunofluorescence was done in all patients. Indirect immunofluorescence using lesional skin as substrate was done in all 25 patients and five patients with other dermatoses. RESULTS: A specific fluorescence pattern corresponding to the distribution of lichen planus specific antigen was observed in the stratum spinosum and granulosum in 22 (88%) patients. It was absent from other parts of the epidermis, dermis and in patients with other dermatoses. CONCLUSION: Indirect immunofluorescence is a useful adjuvant test in lichen planus, particularly in atypical cases.

Characterization of three glycosyltransferases involved in the biosynthesis of the phenolic glycolipid antigens from the Mycobacterium tuberculosis complex.

Mycobacterium tuberculosis and Mycobacterium leprae, the two main mycobacterial pathogens in humans, produce highly specific long chain beta-diols, the dimycocerosates of phthiocerol, and structurally related phenolic glycolipid (PGL) antigens, which are important virulence factors. In addition, M. tuberculosis also secretes glycosylated p-hydroxybenzoic acid methyl esters (p-HBAD) that contain the same carbohydrate moiety as the species-specific PGL of M. tuberculosis (PGL-tb). The genes involved in the biosynthesis of these compounds in M. tuberculosis are grouped on a 70-kilobase chromosomal fragment containing three genes encoding putative glycosyltransferases: Rv2957, Rv2958c, and Rv2962c. To determine the functions of these genes, three recombinant M. tuberculosis strains, in which these genes were individually inactivated, were constructed and biochemically characterized. Our results demonstrated that (i) the biosynthesis of PGL-tb and p-HBAD involves common enzymatic steps, (ii) the Rv2957, Rv2958c, and Rv2962c genes are involved in the formation of the glycosyl moiety of the two classes of molecules, and (iii) the product of Rv2962c catalyzes the transfer of a rhamnosyl residue onto p-hydroxybenzoic acid ethyl ester or phenolphthiocerol dimycocerosates, whereas the products of Rv2958c and Rv2957 add a second rhamnosyl unit and a fucosyl residue to form the species-specific triglycosyl appendage of PGL-tb and p-HBAD. The recombinant strains produced provide the tools to study the role of the carbohydrate domain of PGL-tb and p-HBAD in M. tuberculosis pathogenesis.

CD1a and langerin: acting as more than Langerhans cell markers.

Langerhans cells (LCs) represent a unique DC subset populating the outermost body surface, i.e., the epidermis. Although CD1a and langerin (CD207) are used as specific markers to distinguish LCs from other DC subsets, their immunological functions have remained mostly unknown. A new paper (see the related article beginning on page 701) demonstrates that LCs utilize these markers to induce cellular immune responses to Mycobacterium leprae: CD1a mediates the presentation of nonpeptide antigens to T cells, while langerin facilitates uptake of microbial fragments and perhaps their delivery to a specialized subcellular compartment.

La serología en lepra: últimos avances, potencial, limitaciones y perspectivas: revision del estado del arte

Los anticuerpos específicos pueden ser usados como un marcador indirecto para la carga bacterial en la lepra. Las pruebas para descubrir anticuerpos pueden ser usadas para (i) la clasificación de pacientes por objetivos del tratamiento [la ma yoría de los pacientes multibacilares (MB) son seropositivos, la mayoría de pacientes paucibacilares (PB) no lo son], (ii) para la predicción de un riesgo aumentado de recaída y (iii) la identificación de contactos que presentan un riesgo aumentado de desarrollar lepra. Con la llegada de tests serológicos rápidos, seguros y fáciles de realizar como la prueba del flujo lateral, la aglutinación y técnicas tipo tarjeta, el uso de la seroogy en el campo para estos objetivos adopta una perspectiva factible. De momento presentamos una descripción de los conocimiento actuales y novedades en esta área y se comenta el potencial, limitaciones y las posibles' sutilidades de detección de anticuerpos en la investigación de lepra y su control (AU)

Comparative mycobacterial genomics as a tool for drug target and antigen discovery.

Genomics and the associated downstream technologies are generating vast data sets that provide new opportunities for understanding and combating both infectious and genetic diseases in humans. The genomic approach has been applied to tuberculosis, a major cause of transmissible morbidity and mortality, with notable success. Complete genome sequences are now available for three members of the Mycobacterium tuberculosis complex and the related intracellular pathogen M. leprae. Many of the predictions generated in silico by genomics have been validated through functional analysis, including studies of the transcriptome and proteome, and led to the identification of essential genes. Knowledge of the latter defines potential targets for new and existing drugs and their specificity can be assessed by comparative genomics with the host or other pathogens. Genomics is also furthering tuberculosis vaccine development by pinpointing potentially antigenic proteins as well as providing better diagnostic tools to detect infection.

Evidence for human CD4+ T cells in the CD1-restricted repertoire: derivation of mycobacteria-reactive T cells from leprosy lesions.

Both the CD4-CD8- (double negative) and CD4-CD8+ T cell lineages have been shown to contain T cells which recognize microbial lipid and glycolipid Ags in the context of human CD1 molecules. To determine whether T cells expressing the CD4 coreceptor could recognize Ag in the context of CD1, we derived CD4+ T cell lines from the lesions of leprosy patients. We identified three CD4+ Mycobacterium leprae-reactive, CD1-restricted T cell lines: two CD1b restricted and one CD1c restricted. These T cell lines recognize mycobacterial Ags, one of which has not been previously described for CD1-restricted T cells. The response of CD4+ CD1-restricted T cells, unlike MHC class II-restricted T cells, was not inhibited by anti-CD4 mAb, suggesting that the CD4 coreceptor does not impact positive or negative selection of CD1-restricted T cells. The CD4+ CD1-restricted T cell lines produced IFN-gamma and GM-CSF, the Th1 pattern of cytokines required for cell-mediated immunity against intracellular pathogens, but no detectable IL-4. The existence of CD4+ CD1-restricted T cells that produce a Th1 cytokine pattern suggests a contributory role in immunity to mycobacterial infection.

Pesquisa de antígenos ABH em eritrócitos e na saliva de pacientes hansenianos / ABH antigens research in erythrocytes and at leprosy patients saliva

Os poucos estudos já publicados sobre a determinaçäo de fenótipo secretor dos antígenos ABH na saliva de hansenianos têm demonstrado näo haver uma correlaçäo significativa entre estas substâncias e a suscetibilidade à doença. No presente estudo avaliamos 74 pacientes, sendo 27 virchovianos, 23 tuberculóides e 24 dimorfos, quanto à presença de antíngenos ABH nos eritrócitos e na saliva, pela reaçäo de aglutinaçäo em tubo e inibiçäo de aglutinaçäo. A frequência dos grupos sanguineos ABO e do fenótipo secretor e näo secretor nestes pacientes e no grupo controle foram: O= 40,5(por cento) 49(por cento); AB= 6,8(por cento) 3,6(por cento) e näo secretor= 31,1(por cento) 17,5(por cento). Analisando-se os resultados apresentados nos hansenianos, observamos que näo houve antígenos ABH pesquisados nos eritrócitos e na saliva

Fragmentación en geles de sds-poliacrilamida e inmunotransferencia para la detección de antigenos de una cepa de trypanosoma cruzi aislada en el país y su evaluación con el método de microelisa

Por ser el Trypanosoma cruzi un protozoario de estructura bioquímica compleja presenta en su cubierta celular externa antígenos específicos de su especie y antígenos de reacción cruzada con otros microorganismos. El trypanosoama cruzi comparte el 60 por ciento de sus antígenos solubles con el trypanosoma rangeli,el 30 por ciento con lesihmania donovani y leishmania mexicana y algunos determinantes antigénos con micobacterium tuberculoso. En forma adicional evidencias ecperimentales indican que las cepas de trypanosoma cruzi aisladas a partir de diferentes áreas geográficas de Latinoamérica, se comportan de manera distinta una de otra en el campo inmunológico, clínico y farmacológico. En este estudio demostramos que en el trypanosoma cruzi existen moléculas antigénicamente específicas del parásito, las mismas que al ser utilizadas en el inmunodiagnóstico incrementa la especificiadad de la prueba. En tanro que la sensibilidad mejora al utilizar este antígeno con la técnica de microELISA, la misma que detecta cantidades de anticuerpos en el rango de manogram,os-picogramos. Al realizar el fraccionamiento de las diferentes proteínas estructurales en geles SDS-poliacrilamida de una cepa de trypanosoma cruzi aislada en el país, luego de la subsiguiente inmunotransferencia de esta electroforesis a papel de nitrocelulosa, se logró seleccionar las fracciones antigénicas de 63, 76, 80 y 91 Kd que son reconocidas específicamente por sueros de pacientes con enfermedad de chagas en su fase crónica.Con estos antígenos se estandarizó el inmunodiagnóstico con la prueba de micro elisa. En este inmunoensayo se puede diferenciar con un amplio rango de diferencia las densidades ópticas (D:O) de los sueros provenientes de pacientes chagásicos (D.O 1,797), en relación a los sueros provenientes de pacientes chagásicos (D.O. 1797), en relación a los sueros provenientes de pacientes con leishmaniasis D.O 0.0099),lepra (D.O 0.012), tuberculosis (D.O 0.092), sífilis (D.O 0.067), toxoplasmosis (D.O 0.024). Quedando demostrado la especificidad de las bandas seleccionadas para reconocer sueros de pacientes chagásicos. En forma adicional, en relación a estas moléculas, es importante determinar su naturaleza bioquímica y su papel biológico en la fisiología de trypanonoma cruzi y en la relación huésped-parásito con miras de buscar nuevas estrategias de lucha frente a la enfermedad de chagas.

Detection of S-100 antigen and anti ceramide antibody in sera of leprosy patients with and without reaction.

Levels of anticeramide antibodies and S-100 antigen in leprosy patients with and without reaction are compared in this study. The increase in levels of IgM anti ceramide antibody in the tuberculoid group of patients with reaction, when compared to those without reaction, is significant (P < 0.05). Similarly, significant increase (P < 0.01) was observed in the borderline group with reaction. No significant change in anti ceramide antibody level was observed in the lepromatous group of patients with and without reaction. Mean levels of S-100 were slightly lower in all three groups of patients with reaction, but the differences were not statistically significant.