Montoya AL, Gil ER, Vinales I, Estevao IL, Taboada P, et al.
Carbohydrate research ā¢
The protozoan parasite Leishmania (Viannia) braziliensis is among Latin America's most widespread Leishmania species and is responsible for tegumentary leishmaniasis (TL). This disease has multiple clinical presentations, with cutaneous leishmaniasis (CL) being the most frequent. It manifests as one or a few localized skin ulcers, which can spread to other body areas. Hence, early diagnosis and treatment, typically with pentavalent antimonials, is critical. Traditional diagnostic methods, like parasite culture, microscopy, or the polymerase chain reaction (PCR) for detection of the parasite DNA, have limitations due to the uneven distribution of parasites in biopsy samples. Nonetheless, studies have revealed high levels of parasite-specific anti-Ī±-Gal antibodies in L. (V.) braziliensis-infected patients. Previously, we demonstrated that the neoglycoprotein NGP28b, consisting of the L. (Leishmania) major type-2 glycoinositolphospholipid (GIPL)-3-derived trisaccharide GalpĪ±1,6GalpĪ±1,3GalfĪ² conjugated to bovine serum albumin (BSA) via a linker, acts as a reliable serological biomarker (BMK) for L. (V.) braziliensis infection in Brazil. This indicates the presence of GIPL-3 or a similar structure in this parasite, and its terminal trisaccharide either functions as or is part of an immunodominant glycotope. Here, we explored whether extending the trisaccharide with a mannose unit would enhance its efficacy as a biomarker for the serological detection of L. (V.) braziliensis. We synthesized the tetrasaccharide GalpĪ±1,6GalpĪ±1,3GalfĪ²1,3ManpĪ±(CH)SH (G31) and conjugated it to maleimide-functionalized BSA to afford NGP31b. When we assessed the efficacy of NGP28b and NGP31b by chemiluminescent enzyme-linked immunosorbent assay on a cohort of CL patients with L. (V.) braziliensis infection from Bolivia and Argentina against a healthy control group, both NGPs exhibited similar or identical sensitivity, specificity, and accuracy. This finding implies that the mannose moiety at the reducing end is not part of the glycotope recognized by the parasite-specific anti-Ī±-Gal antibodies in patients' sera, nor does it exert a relevant influence on the terminal trisaccharide's conformation. Moreover, the mannose does not seem to inhibit glycan-antibody interactions. Therefore, NGP31b is a viable and dependable BMK for the serodiagnosis of CL caused by L. (V.) braziliensis.
Montoya AL, Carvajal EG, Ortega-Rodriguez U, Estevao IL, Ashmus RA, et al.
Molecules (Basel, Switzerland) ā¢
Chagas disease (CD) is caused by the parasite and affects 6-7 million people worldwide. The diagnosis is still challenging, due to extensive parasite diversity encompassing seven genotypes (TcI-VI and Tcbat) with diverse ecoepidemiological, biological, and pathological traits. Chemotherapeutic intervention is usually effective but associated with severe adverse events. The development of safer, more effective therapies is hampered by the lack of biomarker(s) (BMKs) for the early assessment of therapeutic outcomes. The mammal-dwelling trypomastigote parasite stage expresses glycosylphosphatidylinositol-anchored mucins (tGPI-MUC), whose -glycans are mostly branched with terminal, nonreducing Ī±-galactopyranosyl (Ī±-Gal) glycotopes. These are absent in humans, and thus highly immunogenic and inducers of specific CD anti-Ī±-Gal antibodies. In search for Ī±-Gal-based BMKs, here we describe the synthesis of neoglycoprotein NGP11b, comprised of a carrier protein decorated with the branched trisaccharide GalĪ±(1,2)[GalĪ±(1,6)]GalĪ². By chemiluminescent immunoassay using sera/plasma from chronic CD (CCD) patients from Venezuela and Mexico and healthy controls, NGP11b exhibited sensitivity and specificity similar to that of tGPI-MUC from genotype TcI, predominant in those countries. Preliminary evaluation of CCD patients subjected to chemotherapy showed a significant reduction in anti-Ī±-Gal antibody reactivity to NGP11b. Our data indicated that NGP11b is a potential BMK for diagnosis and treatment assessment in CCD patients.
Montoya AL, Gil ER, Heydemann EL, Estevao IL, Luna BE, et al.
Molecules (Basel, Switzerland) ā¢
Chagas disease (CD) can be accurately diagnosed by detecting in patients' blood using polymerase chain reaction (PCR). However, parasite-derived biomarkers are of great interest for the serological diagnosis and early evaluation of chemotherapeutic efficacy when PCR may fail, owing to a blood parasite load below the method's limit of detection. Previously, we focused on the detection of specific anti-Ī±-galactopyranosyl (Ī±-Gal) antibodies in chronic CD (CCD) patients elicited by Ī±-Gal glycotopes copiously expressed on insect-derived and mammal-dwelling infective parasite stages. Nevertheless, these stages also abundantly express cell surface glycosylphosphatidylinositol (GPI)-anchored glycoproteins and glycoinositolphospholipids (GIPLs) bearing nonreducing terminal Ī²-galactofuranosyl (Ī²-Gal) residues, which are equally foreign to humans and, therefore, highly immunogenic. Here we report that CCD patients' sera react specifically with synthetic Ī²-Gal-containing glycans. We took a reversed immunoglycomics approach that entailed: (a) Synthesis of GIPL-derived GalĪ²1,3ManĪ±-(CH)SH (glycan ) and GalĪ²1,3ManĪ±1,2-[GalĪ²1,3]ManĪ±-(CH)SH (glycan ); and (b) preparation of neoglycoproteins and , and their evaluation in a chemiluminescent immunoassay. Receiver-operating characteristic analysis revealed that can distinguish CCD sera from sera of healthy individuals with 85.3% sensitivity and 100% specificity. This suggests that GalĪ²1,3ManĪ±1,2-[GalĪ²1,3]ManĪ± is an immunodominant glycotope and that could potentially be used as a novel CCD biomarker.
Viana SM, Montoya AL, Carvalho AM, de MendonƧa BS, Portillo S, et al.
Emerging microbes & infections ā¢
American tegumentary leishmaniasis (TL) caused by is characterized by a spectrum of clinical presentations, ranging from localized cutaneous ulcers (CL), mucosal (ML), or disseminated (DL) disease, to a subclinical (SC) asymptomatic form. Current diagnosis based on parasite culture and/or microscopy lacks sensitivity and specificity. Previous studies showed that patients with CL and ML have very high levels of -specific anti-Ī±-Gal antibodies. However, the native parasite Ī±-Gal glycotope(s) is(are) still elusive, thus they have not yet been explored for a more accurate TL diagnosis. Using a chemiluminescent immunoassay, we evaluated the seroreactivity of TL patients across its clinical spectrum, and of endemic (EC) and nonendemic healthy controls (NEC) against three synthetic neoglycoproteins (NGP29b, NGP30b, and NGP28b), respectively comprising the derived type-2 glycoinositolphospholipid (GIPL)-1 (GalĪ²1,3ManĪ±), GIPL-2 (GalĪ±1,3GalĪ²1,3ManĪ±), and GIPL-3 (GalĪ±1,6GalĪ±1,3GalĪ²) glycotopes. Contrary to NGP29b and NGP30b, NGP28b exhibited high sensitivity and specificity to a CL serum pool. More importantly, NGP28b reacted strongly and specifically with individual sera from distinct clinical forms of TL, especially with SC sera, with 94% sensitivity and 97% specificity, by post-two-graph receiver-operating characteristic curve analysis. Contrary to NGP29b, NGP28b showed low cross-reactivity with Chagas disease and control (NEC/EC) sera. Additionally, seroreactivity of CL patients against NGP28b was significantly decreased after successful chemotherapy, indicating that -specific anti-Ī±-Gal antibodies may serve as an early biomarker of cure in CL. Our data also points towards the applicability of type-2 GIPL-3-derived GalĪ±1,6GalĪ±1,3GalĪ² glycotope for the serological diagnosis of American TL, particularly of the subclinical form.
Montoya AL, Austin VM, Portillo S, Vinales I, Ashmus RA, et al.
JACS Au ā¢
All healthy humans have high levels of natural anti-Ī±-galactosyl (Ī±-Gal) antibodies (elicited by yet uncharacterized glycotopes), which may play important roles in immunoglycomics: (a) potential protection against certain parasitic and viral zoonotic infections; (b) targeting of Ī±-Gal-engineered cancer cells; (c) aiding in tissue repair; and (d) serving as adjuvants in Ī±-Gal-based vaccines. Patients with certain protozoan infections have specific anti-Ī±-Gal antibodies, elicited against parasite-derived Ī±-Gal-bearing glycotopes. These glycotopes, however, remain elusive except for the well-characterized glycotope GalĪ±1,3GalĪ²1,4GlcNAcĪ±, expressed by . The discovery of new parasitic glycotopes is greatly hindered by the enormous structural diversity of cell-surface glycans and the technical challenges of classical immunoglycomics, a top-down approach from cultivated parasites to isolated glycans. Here, we demonstrate that reversed immunoglycomics, a bottom-up approach, can identify parasite species-specific Ī±-Gal-bearing glycotopes by probing synthetic oligosaccharides on neoglycoproteins. This method was tested here seeking to identify as-yet unknown glycotopes specific for , the causative agent of Old-World cutaneous leishmaniasis (OWCL). Neoglycoproteins decorated with synthetic Ī±-Gal-containing oligosaccharides derived from glycoinositolphospholipids served as antigens in a chemiluminescent enzyme-linked immunosorbent assay using sera from OWCL patients and noninfected individuals. Receiver-operating characteristic analysis identified GalĪ±1,3GalĪ² and GalĪ±1,3GalĪ²1,3ManĪ± glycotopes as diagnostic biomarkers for caused OWCL, which can distinguish with 100% specificity from heterologous diseases and caused OWCL. These glycotopes could prove useful in the development of rapid Ī±-Gal-based diagnostics and vaccines for OWCL. Furthermore, this method could help unravel cryptic Ī±-Gal-glycotopes of other protozoan parasites and enterobacteria that elicit the natural human anti-Ī±-Gal antibodies.
Ortega-Rodriguez U, Portillo S, Ashmus RA, Duran JA, Schocker NS, et al.
Methods in molecular biology (Clifton, N.J.) ā¢
Chagas disease (ChD), caused by the protozoan parasite Trypanosoma cruzi, affects millions of people worldwide. Chemotherapy is restricted to two drugs, which are partially effective and may cause severe side effects, leading to cessation of treatment in a significant number of patients. Currently, there are no biomarkers to assess therapeutic efficacy of these drugs in the chronic stage. Moreover, no preventive or therapeutic vaccines are available. In this chapter, we describe the purification of Trypanosoma cruzi trypomastigote-derived glycosylphosphatidylinositol (GPI)-anchored mucins (tGPI-mucins) for their use as antigens for the reliable primary or confirmatory diagnosis and as prognostic biomarkers for early assessment of cure following ChD chemotherapy. We also describe, as an example, the synthesis of a potential tGPI-mucin-derived Ī±-Gal-terminating glycan and its coupling to a carrier protein for use as diagnostic and prognostic biomarker in ChD.
Subramaniam KS, Austin V, Schocker NS, Montoya AL, Anderson MS, et al.
Parasitology ā¢
Outbreaks of Old World cutaneous leishmaniasis (CL) have significantly increased due to the conflicts in the Middle East, with most of the cases occurring in resource-limited areas such as refugee settlements. The standard methods of diagnosis include microscopy and parasite culture, which have several limitations. To address the growing need for a CL diagnostic that can be field applicable, we have identified five candidate neoglycoproteins (NGPs): GalĪ± (NGP3B), GalĪ±(1,3)GalĪ± (NGP17B), GalĪ±(1,3)GalĪ² (NGP9B), GalĪ±(1,6)[GalĪ±(1,2)]GalĪ² (NGP11B), and GalĪ±(1,3)GalĪ²(1,4)GlcĪ² (NGP1B) that are differentially recognized in sera from individuals with Leishmania major infection as compared with sera from heterologous controls. These candidates contain terminal, non-reducing Ī±-galactopyranosyl (Ī±-Gal) residues, which are known potent immunogens to humans. Logistic regression models found that NGP3B retained the best diagnostic potential (area under the curve from receiver-operating characteristic curve = 0.8). Our data add to the growing body of work demonstrating the exploitability of the human anti-Ī±-Gal response in CL diagnosis.
The anaphylaxis response is frequently associated with food allergies, representing a significant public health hazard. Recently, exposure to tick bites and production of specific IgE against Ī±-galactosyl (Ī±-Gal)-containing epitopes has been correlated to red meat allergy. However, this association and the source of terminal, non-reducing Ī±-Gal-containing epitopes have not previously been established in Brazil. Here, we employed the Ī±-1,3-galactosyltransferase knockout mouse (Ī±1,3-GalT-KO) model and bacteriophage QĪ²-virus like particles (QĪ²-VLPs) displaying GalĪ±1,3GalĪ²1,4GlcNAc (GalĪ±3LN) epitopes to investigate the presence of Ī±-Gal-containing epitopes in the saliva of Amblyomma sculptum, a species of the Amblyomma cajennense complex, which represents the main tick that infests humans in Brazil. We confirmed that the Ī±-1,3-galactosyltransferase knockout animals produce significant levels of anti-Ī±-Gal antibodies against the GalĪ±1,3GalĪ²1,4GlcNAc epitopes displayed on QĪ²-virus like particles. The injection of A. sculptum saliva or exposure to feeding ticks was also found to induce both IgG and IgE anti-Ī±-Gal antibodies in Ī±-1,3-galactosyltransferase knockout mice, thus indicating the presence of Ī±-Gal-containing epitopes in the tick saliva. The presence of Ī±-Gal-containing epitopes was confirmed by ELISA and immunoblotting following removal of terminal Ī±-Gal epitopes by Ī±-galactosidase treatment. These results suggest for the first known time that bites from the A. sculptum tick may be associated with the unknown etiology of allergic reactions to red meat in Brazil.
The Ī±-Gal antigen [GalĪ±(1,3)GalĪ²(1,4)GlcNAcĪ±] is an immunodominant epitope displayed by infective trypomastigote forms of Trypanosoma cruzi, the causative agent of Chagas disease. A virus-like particle displaying a high density of Ī±-Gal was found to be a superior reagent for the ELISA-based serological diagnosis of Chagas disease and the assessment of treatment effectiveness. A panel of sera from patients chronically infected with T. cruzi, both untreated and benznidazole-treated, was compared with sera from patients with leishmaniasis and from healthy donors. The nanoparticle-Ī±-Gal construct allowed for perfect discrimination between Chagas patients and the others, avoiding false negative and false positive results obtained with current state-of-the-art reagents. As previously reported with purified Ī±-Gal-containing glycosylphosphatidylinositol-anchored mucins, the current study also showed concentrations of anti-Ī±-Gal IgG to decrease substantially in patients receiving treatment with benznidazole, suggesting that the semiquantitative assessment of serum levels of this highly abundant type of antibody can report on disease status in individual patients.
In the Kingdom of Saudi Arabia (KSA), Old World cutaneous leishmaniasis (CL) is mainly caused by Leishmania major and Leishmania tropica parasites. Diagnosis of CL is predominately made by clinicians, who at times fail to detect the disease and are unable to identify parasite species. Here, we report the development of a chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) to measure the levels of anti-Ī±-galactosyl antibodies in human sera. Using this assay, we have found that individuals infected with either Leishmania spp. had significantly elevated levels (up to 9-fold higher) of anti-Ī±-Gal IgG compared to healthy control individuals. The assay sensitivity was 96% for L. major (95% CI; 94-98%) and 91% for L. tropica (95% CI; 86-98%) infections and therefore equivalent to restriction fragment length polymorphism-polymerase chain reaction analysis of parasite ITS1 gene. In addition, the assay had higher sensitivity than microscopy analysis, which only detected 68 and 45% of the L. major and L. tropica infections, respectively. Interestingly, up to 2 years following confirmed CL cure individuals had 28-fold higher levels of anti-Ī±-Gal IgG compared to healthy volunteers. Monitoring levels of anti-Ī±-Gal antibodies can be exploited as both a diagnostic tool and as a biomarker of cure of Old World CL in disease elimination settings.
Vallejo MC, Matsuo AL, Ganiko L, Medeiros LC, Miranda K, et al.
Eukaryotic cell ā¢
Exosome-like vesicles containing virulence factors, enzymes, and antigens have recently been characterized in fungal pathogens, such as Cryptococcus neoformans and Histoplasma capsulatum. Here, we describe extracellular vesicles carrying highly immunogenic Ī±-linked galactopyranosyl (Ī±-Gal) epitopes in Paracoccidioides brasiliensis. P. brasiliensis is a dimorphic fungus that causes human paracoccidioidomycosis (PCM). For vesicle preparations, cell-free supernatant fluids from yeast cells cultivated in Ham's defined medium-glucose were concentrated in an Amicon ultrafiltration system and ultracentrifuged at 100,000 Ć g. P. brasiliensis antigens were present in preparations from phylogenetically distinct isolates Pb18 and Pb3, as observed in immunoblots revealed with sera from PCM patients. In an enzyme-linked immunosorbent assay (ELISA), vesicle components containing Ī±-Gal epitopes reacted strongly with anti-Ī±-Gal antibodies isolated from both Chagas' disease and PCM patients, with Marasmius oreades agglutinin (MOA) (a lectin that recognizes terminal Ī±-Gal), but only faintly with natural anti-Ī±-Gal. Reactivity was inhibited after treatment with Ī±-galactosidase. Vesicle preparations analyzed by electron microscopy showed vesicular structures of 20 to 200 nm that were labeled both on the surface and in the lumen with MOA. In P. brasiliensis cells, components carrying Ī±-Gal epitopes were found distributed on the cell wall, following a punctuated confocal pattern, and inside large intracellular vacuoles. Lipid-free vesicle fractions reacted with anti-Ī±-Gal in ELISA only when not digested with Ī±-galactosidase, while reactivity with glycoproteins was reduced after Ī²-elimination, which is indicative of partial O-linked chain localization. Our findings open new areas to explore in terms of host-parasite relationships in PCM and the role played in vivo by vesicle components and Ī±-galactosyl epitopes.
