Asymmetric Total Synthesis and Structural Revision of DAT2, an Antigenic Glycolipid from Mycobacterium tuberculosis.

DAT2 is a member of the diacyl trehalose family (DAT) of antigenic glycolipids located in the mycomembrane of Mycobacterium tuberculosis (Mtb). Recently it was shown that the molecular structure of DAT2 had been incorrectly assigned, but the correct structure remained elusive. Herein, the correct molecular structure of DAT2 and its methyl-branched acyl substituent mycolipanolic acid is determined. For this, four different stereoisomers of mycolipanolic acid were prepared in a stereoselective and unified manner, and incorporated into DAT2. A rigorous comparison of the four isomers to the DAT isolated from Mtb H37Rv by NMR, HPLC, GC, and mass spectrometry allowed a structural revision of mycolipanolic acid and DAT2. Activation of the macrophage inducible Ca2+-dependent lectin receptor (Mincle) with all four stereoisomers shows that the natural stereochemistry of mycolipanolic acid / DAT2 provides the strongest activation, which indicates its high antigenicity and potential application in serodiagnostics and vaccine adjuvants.

From the banal to the bizarre: unravelling immune recognition and response to microbial lipids.

Microbes produce a rich array of lipidic species that through their location in the cell wall and ability to mingle with host lipids represent a privileged class of immune-active molecules. Lipid-sensing immunity recognizes microbial lipids from pathogens and commensals causing immune responses. Yet microbial lipids are often heterogeneous, in limited supply and in some cases their structures are incompletely defined. Total synthesis can assist in structural determination, overcome supply issues, and provide access to high-purity, homogeneous samples and analogues. This account highlights synthetic approaches to lipidic species from pathogenic and commensal bacteria and fungi that have supported immunological studies involving lipid sensing through the pattern recognition receptor Mincle and cell-mediated immunity through the CD1-T cell axis.

CD4 and CD8 co-receptors modulate functional avidity of CD1b-restricted T cells.

T cells recognize mycobacterial glycolipid (mycolipid) antigens presented by CD1b molecules, but the role of CD4 and CD8 co-receptors in mycolipid recognition is unknown. Here we show CD1b-mycolipid tetramers reveal a hierarchy in which circulating T cells expressing CD4 or CD8 co-receptor stain with a higher tetramer mean fluorescence intensity than CD4-CD8- T cells. CD4+ primary T cells transduced with mycolipid-specific T cell receptors bind CD1b-mycolipid tetramer with a higher fluorescence intensity than CD8+ primary T cells. The presence of either CD4 or CD8 also decreases the threshold for interferon-γ secretion. Co-receptor expression increases surface expression of CD3ε, suggesting a mechanism for increased tetramer binding and activation. Targeted transcriptional profiling of mycolipid-specific T cells from individuals with active tuberculosis reveals canonical markers associated with cytotoxicity among CD8+ compared to CD4+ T cells. Thus, expression of co-receptors modulates T cell receptor avidity for mycobacterial lipids, leading to in vivo functional diversity during tuberculosis disease.

High Dimensional Immune Profiling Reveals Different Response Patterns in Active and Latent Tuberculosis Following Stimulation With Mycobacterial Glycolipids.

Upon infection with Mycobacterium tuberculosis (Mtb) the host immune response might clear the bacteria, control its growth leading to latent tuberculosis (LTB), or fail to control its growth resulting in active TB (ATB). There is however no clear understanding of the features underlying a more or less effective response. Mtb glycolipids are abundant in the bacterial cell envelope and modulate the immune response to Mtb, but the patterns of response to glycolipids are still underexplored. To identify the CD45+ leukocyte activation landscape induced by Mtb glycolipids in peripheral blood of ATB and LTB, we performed a detailed assessment of the immune response of PBMCs to the Mtb glycolipids lipoarabinomannan (LAM) and its biosynthetic precursor phosphatidyl-inositol mannoside (PIM), and purified-protein derivate (PPD). At 24 h of stimulation, cell profiling and secretome analysis was done using mass cytometry and high-multiplex immunoassay. PIM induced a diverse cytokine response, mainly affecting antigen-presenting cells to produce both pro-inflammatory and anti-inflammatory cytokines, but not IFN-γ, contrasting with PPD that was a strong inducer of IFN-γ. The effect of PIM on the antigen-presenting cells was partly TLR2-dependent. Expansion of monocyte subsets in response to PIM or LAM was reduced primarily in LTB as compared to healthy controls, suggesting a hyporesponsive/tolerance pattern derived from Mtb infection.

Rational design of a hydrolysis-resistant mycobacterial phosphoglycolipid antigen presented by CD1c to T cells.

Whereas proteolytic cleavage is crucial for peptide presentation by classical major histocompatibility complex (MHC) proteins to T cells, glycolipids presented by CD1 molecules are typically presented in an unmodified form. However, the mycobacterial lipid antigen mannosyl-ß1-phosphomycoketide (MPM) may be processed through hydrolysis in antigen presenting cells, forming mannose and phosphomycoketide (PM). To further test the hypothesis that some lipid antigens are processed, and to generate antigens that lead to defined epitopes for future tuberculosis vaccines or diagnostic tests, we aimed to create hydrolysis-resistant MPM variants that retain their antigenicity. Here, we designed and tested three different, versatile synthetic strategies to chemically stabilize MPM analogs. Crystallographic studies of CD1c complexes with these three new MPM analogs showed anchoring of the lipid tail and phosphate group that is highly comparable to nature-identical MPM, with considerable conformational flexibility for the mannose head group. MPM-3, a difluoromethylene-modified version of MPM that is resistant to hydrolysis, showed altered recognition by cells, but not by CD1c proteins, supporting the cellular antigen processing hypothesis. Furthermore, the synthetic analogs elicited T cell responses that were cross-reactive with nature-identical MPM, fulfilling important requirements for future clinical use.

Mycobacterium indicus pranii vaccine immunoprophylaxis in anti-phenolic glycolipid-1-positive leprosy contacts - A pilot study from a tertiary care center in North India.

BACKGROUND: Contacts of leprosy patients have an increased risk of infection with Mycobacterium leprae. Contact tracing and chemo- or immunoprophylaxis are important means of preventing leprosy transmission. AIMS: We aimed to evaluate the efficacy of immunoprophylaxis with Mycobacterium indicus pranii vaccine in reducing anti-phenolic glycolipid-1 titers in household contacts of leprosy patients. METHODS: This prospective single-center study was conducted in a tertiary care center in North India from January 2015 to December 2016. Contacts of leprosy patients (both paucibacillary and multibacillary) were screened for anti-phenolic glycolipid-1 antibodies with enzyme-linked immunosorbent assay. Those found positive were given immunoprophylaxis with a single dose of Mycobacterium indicus pranii vaccine, and anti-phenolic glycolipid-1 titers were evaluated at six and 12 months. All contacts were clinically followed for three years. RESULTS: Of the 135 contacts of 98 leprosy patients that were screened, 128 were recruited. Seventeen of these contacts were positive for anti-phenolic glycolipid-1 antibodies and were given Mycobacterium indicus pranii vaccine. Two contacts were lost to follow-up. After immunoprophylaxis, anti-phenolic glycolipid-1 titers were negative in all patients at all intervals, and no contact developed any clinical signs or symptoms of leprosy during the three-year follow-up. LIMITATIONS: The small number of contacts studied, the short follow-up period and the absence of a control group were limitations of this study. Dicussion: We could not find any papers on natural decline of PGL 1 titres in contacts, although in leprosy patients, these titres may even increase after completion of treatment. However the titres do correlate with bacterial load (reference: Int J Lepr Other Mycobact Dis. 1998 Sep;66(3):356-64) so if the tires decrease or become negative it may be considered as an indirect evidence of bacillary clearance. Hence we may suggest the protective efficacy. Furthermore, as the editor mentioned, considering the small number of positive patients, a control group was not possible in the present pilot study, but such studies may be carried out in the future. CONCLUSION: Immunoprophylaxis with Mycobacterium indicus pranii vaccine is effective and safe in preventing disease in contacts of leprosy patients. However, these findings need to be replicated in larger studies.

Latent leprosy infection identified by dual RLEP and anti-PGL-I positivity: Implications for new control strategies.

The number of new cases of leprosy reported worldwide has remained essentially unchanged for the last decade despite continued global use of free multidrug therapy (MDT) provided to any diagnosed leprosy patient. In order to more effectively interrupt the chain of transmission, new strategies will be required to detect those with latent disease who contribute to furthering transmission. To improve the ability to diagnose leprosy earlier in asymptomatic infected individuals, we examined the combined use of two well-known biomarkers of M. leprae infection, namely the presence of M. leprae DNA by PCR from earlobe slit skin smears (SSS) and positive antibody titers to the M. leprae-specific antigen, Phenolic Glycolipid I (anti-PGL-I) from leprosy patients and household contacts living in seven hyperendemic cities in the northern state of Pará, Brazilian Amazon. Combining both tests increased sensitivity, specificity and accuracy over either test alone. A total of 466 individuals were evaluated, including 87 newly diagnosed leprosy patients, 52 post-treated patients, 296 household contacts and 31 healthy endemic controls. The highest frequency of double positives (PGL-I+/RLEP+) were detected in the new case group (40/87, 46%) with lower numbers for treated (12/52, 23.1%), household contacts (46/296, 15.5%) and healthy endemic controls (0/31, 0%). The frequencies in these groups were reversed for double negatives (PGL-I-/RLEP-) for new cases (6/87, 6.9%), treated leprosy cases (15/52, 28.8%) and the highest in household contacts (108/296, 36.5%) and healthy endemic controls (24/31, 77.4%). The data strongly suggest that household contacts that are double positive have latent disease, are likely contributing to shedding and transmission of disease to their close contacts and are at the highest risk of progressing to clinical disease. Proposed strategies to reduce leprosy transmission in highly endemic areas may include chemoprophylactic treatment of this group of individuals to stop the spread of bacilli to eventually lower new case detection rates in these areas.

Breast Milk: A Source of Functional Compounds with Potential Application in Nutrition and Therapy.

Breast milk is an unbeatable food that covers all the nutritional requirements of an infant in its different stages of growth up to six months after birth. In addition, breastfeeding benefits both maternal and child health. Increasing knowledge has been acquired regarding the composition of breast milk. Epidemiological studies and epigenetics allow us to understand the possible lifelong effects of breastfeeding. In this review we have compiled some of the components with clear functional activity that are present in human milk and the processes through which they promote infant development and maturation as well as modulate immunity. Milk fat globule membrane, proteins, oligosaccharides, growth factors, milk exosomes, or microorganisms are functional components to use in infant formulas, any other food products, nutritional supplements, nutraceuticals, or even for the development of new clinical therapies. The clinical evaluation of these compounds and their commercial exploitation are limited by the difficulty of isolating and producing them on an adequate scale. In this work we focus on the compounds produced using milk components from other species such as bovine, transgenic cattle capable of expressing components of human breast milk or microbial culture engineering.

T Cells Specific for a Mycobacterial Glycolipid Expand after Intravenous Bacillus Calmette-Guérin Vaccination.

Intradermal vaccination with Mycobacterium bovis bacillus Calmette-Guérin (BCG) protects infants from disseminated tuberculosis, and i.v. BCG protects nonhuman primates (NHP) against pulmonary and extrapulmonary tuberculosis. In humans and NHP, protection is thought to be mediated by T cells, which typically recognize bacterial peptide Ags bound to MHC proteins. However, during vertebrate evolution, T cells acquired the capacity to recognize lipid Ags bound to CD1a, CD1b, and CD1c proteins expressed on APCs. It is unknown whether BCG induces T cell immunity to mycobacterial lipids and whether CD1-restricted T cells are resident in the lung. In this study, we developed and validated Macaca mulatta (Mamu) CD1b and CD1c tetramers to probe ex vivo phenotypes and functions of T cells specific for glucose monomycolate (GMM), an immunodominant mycobacterial lipid Ag. We discovered that CD1b and CD1c present GMM to T cells in both humans and NHP. We show that GMM-specific T cells are expanded in rhesus macaque blood 4 wk after i.v. BCG, which has been shown to protect NHP with near-sterilizing efficacy upon M. tuberculosis challenge. After vaccination, these T cells are detected at high frequency within bronchoalveolar fluid and express CD69 and CD103, markers associated with resident memory T cells. Thus, our data expand the repertoire of T cells known to be induced by whole cell mycobacterial vaccines, such as BCG, and show that lipid Ag-specific T cells are resident in the lungs, where they may contribute to protective immunity.

Antiglycolipid antibodies in Guillain-Barré and Fisher syndromes: discovery, current status and future perspective.

Guillain-Barré syndrome (GBS) and Fisher syndrome (FS) are acute autoimmune neuropathies, often preceded by an infection. Antiglycolipid antibody titres are frequently elevated in sera from the acute-phase patients. Particularly, IgG anti-GQ1b antibodies are positive in as high as 90% of FS cases and thus useful for diagnosis. The development of animal models of antiglycolipid antibody-mediated neuropathies proved that some of these antibodies are directly involved in the pathogenetic mechanisms by binding to the regions where the respective target glycolipid is specifically localised. Discovery of the presence of the antibodies that specifically recognise a new conformational epitope formed by two different gangliosides (ganglioside complex) in the acute-phase sera of some patients with GBS suggested the carbohydrate-carbohydrate interaction between glycolipids. This finding indicated the need for further research in basic glycobiological science. Antiglycolipid antibodies, in particular antigangliosides antibodies, are mostly detected in acute motor axonal neuropathy type of GBS and in FS, and less frequently in the acute inflammatory demyelinating polyneuropathy (AIDP) type of GBS or in central nervous system (CNS) diseases. In the future, the search for the putative antibodies in AIDP and those that might be present in CNS diseases should continue. In addition, more efficient standardisation of antiglycolipid antibody detection methods and use as biomarkers in daily clinical practice in neurology is needed.

Trafficking of Mycobacterium tuberculosis Envelope Components and Release Within Extracellular Vesicles: Host-Pathogen Interactions Beyond the Wall.

Components of Mycobacterium tuberculosis (Mtb) envelope such as lipoproteins, lypoglycans, lipids, and glycolipids act as Pathogen Associated Molecular Patterns and/or antigens, hence contributing in different ways to the bacillus recognition, phagocytosis, and to immune responses modulation. However, Mtb envelope components are not only encountered at the bacillus-host direct contact but can act remotely from the bacillus envelope. Indeed, they are also released from the bacillus envelope and are detected in different compartments such as the infected cells endosomal compartments or in extracellular vesicles produced by the bacillus itself or by infected cells. Characterizing their trafficking is of main importance for our understanding of their role in host-pathogen interactions and consequently for their potential use as vaccine components. This review aims at providing an overview of the current knowledge of the nature of Mtb envelope components shuttled within extracellular vesicles, the interaction of these vesicles with host immune cells and the remaining black holes.

Cell wall glycolipids from Corynebacterium pseudotuberculosis strains with different virulences differ in terms of composition and immune recognition.

Caseous lymphadenitis (CLA) is an infectious disease caused by Corynebacterium pseudotuberculosis in small ruminants and is characterized by the development of granulomas in the lymph nodes, spleen, liver, and lungs. Although little is known about the host-pathogen relationship of this bacterium, it was previously reported that the pathogen's lipids are important for its taxonomic classification and survival inside macrophages. However, there are no studies regarding the composition of these molecules. In this study, cell wall glycolipids from two C. pseudotuberculosis strains presenting different virulence profiles were purified and its composition was characterized. A difference was observed between the electrophoretic and chromatogram profiles for cell wall components from the two strains, mainly among molecules with low molecular weights. IgM from sheep with acute CLA recognized antigens with an estimated molecular weight of 11 kDa of the low-pathogenicity strain, while low-molecular weight antigens from the high-pathogenicity strain presented a lower recognition by these antibodies. Mass spectrometry analysis showed that the cell wall of the high-pathogenicity strain contained glycolipids with high amounts of unsaturated fatty acids and glycerophosphoinositols, which may contribute to the capacity of this strain to cause severe disease. In conclusion, it is indicated that cell wall non-protein antigens can play a key role in C. pseudotuberculosis virulence.

Glycolipid-peptide vaccination induces liver-resident memory CD8+ T cells that protect against rodent malaria.

Liver resident-memory CD8+ T cells (TRM cells) can kill liver-stage Plasmodium-infected cells and prevent malaria, but simple vaccines for generating this important immune population are lacking. Here, we report the development of a fully synthetic self-adjuvanting glycolipid-peptide conjugate vaccine designed to efficiently induce liver TRM cells. Upon cleavage in vivo, the glycolipid-peptide conjugate vaccine releases an MHC I-restricted peptide epitope (to stimulate Plasmodium-specific CD8+ T cells) and an adjuvant component, the NKT cell agonist α-galactosylceramide (α-GalCer). A single dose of this vaccine in mice induced substantial numbers of intrahepatic malaria-specific CD8+ T cells expressing canonical markers of liver TRM cells (CD69, CXCR6, and CD101), and these cells could be further increased in number upon vaccine boosting. We show that modifications to the peptide, such as addition of proteasomal-cleavage sequences or epitope-flanking sequences, or the use of alternative conjugation methods to link the peptide to the glycolipid improved liver TRM cell generation and led to the development of a vaccine able to induce sterile protection in C57BL/6 mice against Plasmodium berghei sporozoite challenge after a single dose. Furthermore, this vaccine induced endogenous liver TRM cells that were long-lived (half-life of ~425 days) and were able to maintain >90% sterile protection to day 200. Our findings describe an ideal synthetic vaccine platform for generating large numbers of liver TRM cells for effective control of liver-stage malaria and, potentially, a variety of other hepatotropic infections.

Serum immunoglobulin a antibodies to glycopeptidolipid core antigen for Mycobacteroides abscessus complex lung disease.

Background: Mycobacteroides abscessus complex (MABC) exhibits smooth morphotypes, expressing glycopeptidolipid (GPL), and rough morphotypes, expressing diminished GPL, on the MABC cell wall. Few reports have focused on the relationship between anti-GPL-core immunoglobulin A (IgA) antibody and colony morphology in MABC lung disease. Methods: This study aimed to test GPL core antigen in patients with MABC lung disease to investigate the relationship between coinfection/contamination in other nontuberculous mycobacteria species and colony morphology variant in MABC isolates. Patients with MABC lung disease and contamination diagnosed between 2012 and 2017 at our hospital were enrolled retrospectively. Results: Of the assessed patients, 43 patients with MABC lung disease and 13 with MABC contamination were included. There was a significant difference in anti-GPL-core IgA antibody levels between them (P = 0.02). Forty-three patients with MABC lung disease were divided into two groups as positive and negative antibodies groups. A significant increase in the positive anti-GPL-core IgA antibody was observed in coexistence with both Mycobacterium avium complex (MAC) (P = 0.02) and the isolate of the smooth variant (P = 0.03) in MABC. Conclusions: Anti-GPL-core IgA antibodies in patients with MABC are greatly influenced by MAC coexistence, and colony morphology variant of the MABC isolate.

Patient experience with subcutaneous immunoglobulin 20%, Ig20Gly, for primary immunodeficiency diseases: a prespecified post hoc analysis of combined data from 2 pivotal trials.

BACKGROUND: Often, patients with primary immunodeficiency diseases (PID), which are marked by the absence or loss of functional antibodies, require lifelong treatment with immunoglobulin (IG) replacement therapy administered either intravenously (intravenous immunoglobulin [IVIG]) or subcutaneously (subcutaneous immunoglobulin [SCIG]). In patients with PID, the 20% SCIG product, Ig20Gly, was shown to be efficacious and well tolerated in 2 phase 2/3 trials conducted in North America and Europe. This analysis evaluated patient satisfaction with Ig20Gly therapy and treatment preferences. METHODS: This prespecified post hoc analysis showed combined data from 2 Ig20Gly pivotal trials. Treatment satisfaction was assessed in the pre-Ig20Gly period and after ≥11 months of Ig20Gly treatment using the Life Quality Index (LQI; both studies) and the Treatment Satisfaction Questionnaire for Medication-9 (TSQM-9; North American study only). Treatment preference was assessed using a survey at the end of the European study. Median within-patient differences in LQI and TSQM-9 scores between the pre-Ig20Gly period and the end of the Ig20Gly treatment period were assessed using the Wilcoxon signed-rank test. RESULTS: A total of 113 patients (n = 68 [North American]; n = 45 [Europe]) with PID were included in the analysis. In the combined LQI analysis (n = 110), significant improvements were observed in the treatment interference (median ∆: + 2.8; P = 0.006) and therapy setting (median ∆: + 5.6; P < 0.0001) domains, and in the item-level scores for convenience (median ∆: + 1.0; P < 0.0001) and interference with work/school (median ∆: + 1.0; P = 0.0001) categories. In the subgroup analyses, significant improvements in the treatment interference and therapy setting domains and the convenience and interference with work/school items were observed for those who had previously received treatment outside the home, those who had previously received IVIG, and those in the North American study. Significant improvements were observed in the TSQM-9 treatment convenience domain (median ∆: + 11.1; P < 0.0001) and selected item-level scores in the North American study. In the European study, most (88.9%) patients preferred to continue Ig20Gly versus other IG treatments. CONCLUSIONS: After ≥11 months of taking Ig20Gly, patients reported high levels of treatment satisfaction, convenience, and preference for Ig20Gly, with consistent results across studies and use of multiple patient-reported outcome measures.

[The use of synthetic glycoconjugates as components of the immunochromatographic test for rapid serological diagnosis of leprosy.]

The glycoconjugates with BSA (bovine serum albumin) were synthesized using a next saccharide: disaccharide derivative M.leprae PGL-1 (phenolic glycolipid-1); a complex of the disaccharide fragment and the branched hexasaccharide fragment LAM (lipoarabinomannan); diarabinofuranose fragment LAM. These glycoconjugates were used as antigenic components for leprosy rapid serotest construction in immunochromatographic format (leprosy LF serotest). The data obtained with sera of leprosy patients, patients who have been in contact with leprosy, and healthy donors indicate that the most promising antigenic component is a BSA conjugate with two synthetic epitopes - a disaccharide derivative of PGL-1 and a branched hexasaccharide fragment of LAM. The leprosy LF serotest with such glycoconjugate demonstrated the greatest diagnostic sensitivity for main forms of leprosy - paucibacillary (PB) and multibacillary (MB).

Navigating the Role of CD1d/Invariant Natural Killer T-cell/Glycolipid Immune Axis in Multiple Myeloma Evolution: Therapeutic Implications.

Multiple myeloma (MM) is an incurable B-cell malignancy. The immunotherapeutic approach for MM therapy is evolving. The Cd1d/invariant natural killer T-cell/glycolipid immune axis belongs to the innate immunity, and we have highlighted role in myeloma pathogenesis in the present study. The recent development of the chimeric antigen receptor (CAR19)-invariant natural killer T-cells resulted in our renewed interest in this immune system and offer new perspectives for future anti-MM immunotherapies.

Immunology of carbohydrate-based vaccines.

Carbohydrates are considered as promising targets for vaccine development against infectious diseases where cell surface glycan's on many infectious agents are attributed to playing an important role in pathogenesis. Understanding the relationship between carbohydrates and immune components at a molecular level is crucial for the development of well-defined vaccines. Recently, carbohydrate immunology research has been accelerated by the development of new technologies that contribute to the design of optimum antigens, synthesis of antigens and the studies of antigen-antibody interactions, and as a result, several promising carbohydrate-based vaccine candidates have been prepared in recent years. This article briefly presents the mechanistic pathways of polysaccharide, glycoconjugate, glycolipid and zwitterionic vaccines and the interplay between carbohydrate antigen and immune response.