The immunologic abnormalities in patients with paroxysmal nocturnal hemoglobinuria are associated with disease progression.

OBJECTIVES: To suggest the presence of a hyperimmune state in patients, and indicate that immune system attack on glycosylphosphatidylinositol (+) (GPI+) cells while escaping GPI- cell immunity. METHODS: We retrospective the immune cell subtypes in peripheral blood from 25 patients visiting Tianjin Medical University General Hospital, Tianjin, China, with classical paroxysmal nocturnal hemoglobinuria (PNH) and 50 healthy controls. RESULTS: The total CD3+ and CD3+CD8+ cell levels were higher in patients with PNH. The CD3+ cells are positively, correlated with lactate dehydrogenase (LDH; r=0.5453, p=0.0040), indirect bilirubin (r=0.4260, p=0.0379) and Flear- cells in monocytes (r=0.4099, p=0.0303). However, a negative correlation was observed between CD3+ cells and hemoglobin (r= -0.4530, p=0.0105). The total CD19+ cells decreased in patients, and CD19+ cells were negatively correlated with LDH (r= -0.5640, p=0.0077) and Flear- cells in monocytes (r= -0.4432, p=0.0341). Patients showed an increased proportion of total dendritic cells (DCs), with a higher proportion of myeloid DCs (mDCs) within the DC population. Moreover, the proportion of mDC/DC was positively correlated with CD59- cells (II + III types) in red cells (r=0.7941, p=0.0004), Flear- cells in granulocytes (r=0.5357, p=0.0396), and monocytes (r=0.6445, p=0.0095). CONCLUSION: Our results demonstrated that immune abnormalities are associated with PNH development.

Neospora caninum glycosylphosphatidylinositols used as adjuvants modulate cellular immune responses induced in vitro by a nanoparticle-based vaccine.

Neospora caninum causes abortion in ruminants, leading to important economic losses and no efficient treatment or vaccine against neosporosis is available. Considering the complexity of the strategies developed by intracellular apicomplexan parasites to escape immune system, future vaccine formulations should associate the largest panel of antigens and adjuvants able to better stimulate immune responses than natural infection. A mucosal vaccine, constituted of di-palmitoyl phosphatidyl glycerol-loaded nanoparticles (DGNP) and total extract (TE) of soluble antigens of Toxoplasma gondii, has demonstrated its efficacy, decreasing drastically the parasite burden. Here, DGNP were loaded with N. caninum TE and glycosylphosphatidylinositol (GPI) of N. caninum as Toll-like receptor (TLR) adjuvant able to induce specific cellular and humoral immune responses. Activation of TLR2 and TLR4 signalling pathway in HEK reporter cells induced by GPI was abrogated after its incorporation into DGNP. However, in murine bone marrow-derived dendritic cells, an adjuvant effect of GPI was observed with higher levels of interleukin (IL)-1ß, reduced levels of IL-6, IL-12p40 and IL-10, and decreased expression of major histocompatibility complex (MHC) molecules. GPI also modulated the responses of bovine peripheral blood mononuclear cells, by increasing the production of IFN-γ and by decreasing the expression of MHC molecules. Altogether, these results suggest that GPI delivered by the DGNP might modulate cell responses through the activation of an intracellular pathway of signalisation in a TLR-independent manner. In vivo experiments are needed to confirm the potent adjuvant properties of N. caninum GPI in a vaccine strategy against neosporosis.

Babesia divergens glycosylphosphatidylinositols modulate blood coagulation and induce Th2-biased cytokine profiles in antigen presenting cells.

Glycosylphosphatidylinositols (GPIs) are glycolipids described as toxins of protozoan parasites due to their inflammatory properties in mammalian hosts characterized by the production of interleukin (IL)-1, IL-12 and tumor necrosis factor (TNF)-α. In the present work, we studied the cytokines produced by antigen presenting cells in response to ten different GPI species extracted from Babesia divergens, responsible for babesiosis. Interestingly, B. divergens GPIs induced the production of anti-inflammatory cytokines (IL-2, IL-5) and of the regulatory cytokine IL-10 by macrophages and dendritic cells. In contrast to all protozoan GPIs studied until now, GPIs from B. divergens did not stimulate the production of TNF-α and IL-12, leading to a unique Th1/Th2 profile. Analysis of the carbohydrate composition of the B. divergens GPIs indicated that the di-mannose structure was different from the evolutionary conserved tri-mannose structure, which might explain the particular cytokine profile they induce. Expression of major histocompatibility complex (MHC) molecules on dendritic cells and apoptosis of mouse peritoneal cells were also analysed. B. divergens GPIs did not change expression of MHC class I, but decreased expression of MHC class II at the cell surface, while GPIs slightly increased the percentages of apoptotic cells. During pathogenesis of babesiosis, the inflammation-coagulation auto-amplification loop can lead to thrombosis and the effect of GPIs on coagulation parameters was investigated. Incubation of B. divergens GPIs with rat plasma ex vivo led to increase of fibrinogen levels and to prolonged activated partial thromboplastin time, suggesting a direct modulation of the extrinsic coagulation pathway by GPIs.

Vaccine-Induced Carbohydrate-Specific Memory B Cells Reactivate During Rodent Malaria Infection.

A long-standing challenge in malaria is the limited understanding of B cell immunity, previously hampered by lack of tools to phenotype rare antigen-specific cells. Our aim was to develop a method for identifying carbohydrate-specific B cells within lymphocyte populations and to determine whether a candidate vaccine generated functional memory B cells (MBCs) that reactivated upon challenge with Plasmodium (pRBCs). To this end, a new flow cytometric probe was validated and used to determine the kinetics of B cell activation against the candidate vaccine glycosylphosphatidylinositol conjugated to Keyhole Limpet Haemocyanin (GPI-KLH). Additionally, immunized C57BL/6 mice were rested (10 weeks) and challenged with pRBCs or GPI-KLH to assess memory B cell recall against foreign antigen. We found that GPI-specific B cells were detectable in GPI-KLH vaccinated mice, but not in Plasmodium-infected mice. Additionally, in previously vaccinated mice GPI-specific IgG1 MBCs were reactivated against both pRBCs and synthetic GPI-KLH, which resulted in increased serum levels of anti-GPI IgG in both challenge approaches. Collectively our findings contribute to the understanding of B cell immunity in malaria and have important clinical implications for inclusion of carbohydrate conjugates in malaria vaccines.

Detection of Anti-Toxoplasma gondii Antibodies in Human Sera Using Synthetic Glycosylphosphatidylinositol Glycans on a Bead-Based Multiplex Assay.

Toxoplasmosis, while often an asymptomatic parasitic disease in healthy individuals, can cause severe complications in immunocompromised persons and during pregnancy. The most common method to diagnose Toxoplasma gondii infections is the serological determination of antibodies directed against parasite protein antigens. Here we report the use of a bead-based multiplex assay containing a synthetic phosphoglycan portion of the Toxoplasma gondii glycosylphosphatidylinositol (GPI1) for the detection of GPI1-specific antibodies in human sera. The glycan was conjugated to beads at the lipid site to retain its natural orientation and its immunogenic groups. We compared the response against GPI1 with that against the protein antigen SAG1, a common component of commercial serological assays, via the detection of parasite-specific human IgG and IgM antibodies, respectively. The GPI1-based test is in excellent agreement with the results for the commercial ELISA, as the ROC analysis of the GPI1 test shows 97% specificity and 98% sensitivity for the assay. GPI1 was a more reliable predictor for a parasite-specific IgM response compared to SAG1, indicating that a bead-based multiplex assay using GPI1 in combination with SAG1 may strengthen Toxoplasma gondii serology, in particular in seroepidemiological studies.

Staphylococcal phosphatidylinositol-specific phospholipase C potentiates lung injury via complement sensitisation.

Staphylococcus aureus is frequently isolated from patients with community-acquired pneumonia and acute respiratory distress syndrome (ARDS). ARDS is associated with staphylococcal phosphatidylinositol-specific phospholipase C (PI-PLC); however, the role of PI-PLC in the pathogenesis and progression of ARDS remains unknown. Here, we showed that recombinant staphylococcal PI-PLC possesses enzyme activity that causes shedding of glycosylphosphatidylinositol-anchored CD55 and CD59 from human umbilical vein endothelial cell surfaces and triggers cell lysis via complement activity. Intranasal infection with PI-PLC-positive S. aureus resulted in greater neutrophil infiltration and increased pulmonary oedema compared with a plc-isogenic mutant. Although indistinguishable proinflammatory genes were induced, the wild-type strain activated higher levels of C5a in lung tissue accompanied by elevated albumin instillation and increased lactate dehydrogenase release in bronchoalveolar lavage fluid compared with the plc- mutant. Following treatment with cobra venom factor to deplete complement, the wild-type strain with PI-PLC showed a reduced ability to trigger pulmonary permeability and tissue damage. PI-PLC-positive S. aureus induced the formation of membrane attack complex, mainly on type II pneumocytes, and reduced the level of CD55/CD59, indicating the importance of complement regulation in pulmonary injury. In conclusion, S. aureus PI-PLC sensitised tissue to complement activation leading to more severe tissue damage, increased pulmonary oedema, and ARDS progression.

Early suppression of B cell immune responses by low doses of chloroquine and pyrimethamine: implications for studying immunity in malaria.

Malaria remains a significant worldwide public health problem. To address biological questions, researchers rely on the experimental murine model. For decades, chloroquine (CQ) and pyrimethamine (Pyr) have been used to clear Plasmodium infections in experimental animals using standardised accepted protocols and, because of this, drug-treated controls are rarely included. However, there is limited data available on the modulation of anti-malarial immunity, including generation of memory B cells, when these drugs are administered days after malaria infection. We investigated B cell responses to an important malaria glycolipid, glycosylphosphatidylinositol (GPI), and the hapten nitrophenol (NP), with or without standard CQ and Pyr treatment using the murine model. At day 14, CQ/Pyr treatment significantly suppressed the frequency of NP+IgG1+ memory B cells in NP-KLH-immunised mice. Furthermore, CQ/Pyr-treated NP-KLH-immunised mice did not have significantly higher cellular counts of NP+ B cells, germinal centre B cells, nor NP+IgG1+ memory B cells than naïve mice (CQ/Pyr treated and untreated). CQ/Pyr-treated GPI-KLH-immunised mice did not have significantly higher cellular counts of GPI+ B cells than naïve untreated mice. By day 28, this effect appeared to resolve since all immunised mice, whether treated or untreated, had significantly higher B cell proliferative responses than naïve mice (CQ/Pyr treated and untreated) for the majority of B cell phenotypes. The current study emphasises the potential for drug modulation of antigenic B cell responses when using standardised malaria treatment protocols in the experimental murine model. It is recommended that drug-treated controls are included when using experimental malaria infections to address biological questions.

Isolation and purification of glycosylphosphatidylinositols (GPIs) in the schizont stage of Theileria annulata and determination of antibody response to GPI anchors in vaccinated and infected animals.

BACKGROUND: Tropical theileriosis is widely distributed from North Africa to East Asia. It is a tick-borne disease caused by Theileria annulata, an obligate two-host intracellular protozoan parasite of cattle. Theileria annulata use leukocytes and red blood cells for completion of the life-cycle in mammalian hosts. The stage of Theileria annulata in monocytes and B lymphocytes of cattle is an important step in pathogenicity and diagnosis of the disease. Glycosylphosphatidylinositols (GPIs) are a distinct class of glycolipid structures found in eukaryotic cells and are implicated in several biological functions. GPIs are particularly abundant in protozoan parasites, where they are found as free glycolipids or attached to proteins in the plasma membrane. RESULTS: In this study we first isolated and purified schizonts of Theileria annulata from infected leukocytes in Theileria annulata vaccine cell line (S15) by aerolysin-percoll technique. Then, the free GPIs of schizont stage and isolated GPI from cell membrane glycoproteins were purified by high performance liquid chromatography (HPLC) and confirmed by gas chromatography-mass spectrometry (GC-MS). Furthermore, enzyme linked immunosorbent assay (ELISA) on the serum samples obtained from naturally infected, as well as Theileria annulata-vaccinated animals, confirmed a significant (P < 0.01) high level of anti-GPI antibody in their serum. CONCLUSIONS: The results presented in this study show, to our knowledge for the first time, the isolation of GPI from the schizont stage of Theileria annulata and demonstrate the presence of anti-GPI antibody in the serum of naturally infected as well as vaccinated animals. This finding is likely to be valuable in studies aimed at the evaluation of chemically structures of GPIs in the schizont stage of Theileria annulata and also for pathogenicity and immunogenicity studies with the aim to develop GPI-based therapies or vaccines.

Malaria Derived Glycosylphosphatidylinositol Anchor Enhances Anti-Pfs25 Functional Antibodies That Block Malaria Transmission.

Malaria, one of the most common vector borne human diseases, is a major world health issue. In 2015 alone, more than 200 million people were infected with malaria, out of which, 429 000 died. Even though artemisinin-based combination therapies (ACT) are highly effective at treating malaria infections, novel efforts toward development of vaccines to prevent transmission are still needed. Pfs25, a postfertilization stage parasite surface antigen, is a leading transmission-blocking vaccine (TBV) candidate. It is postulated that Pfs25 anchors to the cell membrane using a glycosylphosphatidylinositol (GPI) linker, which itself possesses pro-inflammatory properties. In this study, Escherichia coli derived extract (XtractCF+TM) was used in cell free protein synthesis [CFPS] to successfully express >200 mg/L of recombinant Pfs25 with a C-terminal non-natural amino acid (nnAA), namely, p-azidomethyl phenylalanine (pAMF), which possesses a reactive azide group. Thereafter, a unique conjugate vaccine (CV), namely, Pfs25-GPI was generated with dibenzocyclooctyne (DBCO) derivatized glycan core of malaria GPI using a simple but highly efficient copper free click chemistry reaction. In mice immunized with Pfs25 or Pfs25-GPI, the Pfs25-GPI group showed significantly higher titers compared to the Pfs25 group. Moreover, only purified IgGs from Pfs25-GPI group were able to significantly block transmission of parasites to mosquitoes, as judged by a standard membrane feeding assay [SMFA]. To our knowledge, this is the first report of the generation of a CV using Pfs25 and malaria specific GPI where the GPI is shown to enhance the ability of Pfs25 to elicit transmission blocking antibodies.

IgG antibodies to synthetic GPI are biomarkers of immune-status to both Plasmodium falciparum and Plasmodium vivax malaria in young children.

BACKGROUND: Further reduction in malaria prevalence and its eventual elimination would be greatly facilitated by the development of biomarkers of exposure and/or acquired immunity to malaria, as well as the deployment of effective vaccines against Plasmodium falciparum and Plasmodium vivax. A better understanding of the acquisition of immunity in naturally-exposed populations is essential for the identification of antigens useful as biomarkers, as well as to inform rational vaccine development. METHODS: ELISA was used to measure total IgG to a synthetic form of glycosylphosphatidylinositol from P. falciparum (PfGPI) in a cohort of 1-3 years old Papua New Guinea children with well-characterized individual differences in exposure to P. falciparum and P. vivax blood-stage infections. The relationship between IgG levels to PfGPI and measures of recent and past exposure to P. falciparum and P. vivax infections was investigated, as well as the association between antibody levels and prospective risk of clinical malaria over 16 months of follow-up. RESULTS: Total IgG levels to PfGPI were low in the young children tested. Antibody levels were higher in the presence of P. falciparum or P. vivax infections, but short-lived. High IgG levels were associated with higher risk of P. falciparum malaria (IRR 1.33-1.66, P = 0.008-0.027), suggesting that they are biomarkers of increased exposure to P. falciparum infections. Given the cross-reactive nature of antibodies to PfGPI, high IgG levels were also associated with reduced risk of P. vivax malaria (IRR 0.65-0.67, P = 0.039-0.044), indicating that these antibodies are also markers of acquired immunity to P. vivax. CONCLUSIONS: This study highlights that in young children, IgG to PfGPI might be a useful marker of immune-status to both P. falciparum and P. vivax infections, and potentially useful to help malaria control programs to identify populations at-risk. Further functional studies are necessary to confirm the potential of PfGPI as a target for vaccine development.

Parasite Carbohydrate Vaccines.

Vaccination is an efficient means of combating infectious disease burden globally. However, routine vaccines for the world's major human parasitic diseases do not yet exist. Vaccines based on carbohydrate antigens are a viable option for parasite vaccine development, given the proven success of carbohydrate vaccines to combat bacterial infections. We will review the key components of carbohydrate vaccines that have remained largely consistent since their inception, and the success of bacterial carbohydrate vaccines. We will then explore the latest developments for both traditional and non-traditional carbohydrate vaccine approaches for three of the world's major protozoan parasitic diseases-malaria, toxoplasmosis, and leishmaniasis. The traditional prophylactic carbohydrate vaccine strategy is being explored for malaria. However, given that parasite disease biology is complex and often arises from host immune responses to parasite antigens, carbohydrate vaccines against deleterious immune responses in host-parasite interactions are also being explored. In particular, the highly abundant glycosylphosphatidylinositol molecules specific for Plasmodium, Toxoplasma, and Leishmania spp. are considered exploitable antigens for this non-traditional vaccine approach. Discussion will revolve around the application of these protozoan carbohydrate antigens for vaccines currently in preclinical development.

Interaction between Plasmodium Glycosylphosphatidylinositol and the Host Protein Moesin Has No Implication in Malaria Pathology.

Glycosylphosphatidylinositol (GPI) anchor of Plasmodium falciparum origin is considered an important toxin leading to severe malaria pathology through stimulation of pro-inflammatory responses from innate immune cells. Even though the GPI-induced immune response is widely described to be mediated by pattern recognition receptors such as TLR2 and TLR4, previous studies have revealed that these two receptors are dispensable for the development of severe malaria pathology. Therefore, this study aimed at the identification of potential alternative Plasmodium GPI receptors. Herein, we have identified the host protein moesin as an interaction partner of Plasmodium GPI in vitro. Given previous reports indicating the relevance of moesin especially in the LPS-mediated induction of pro-inflammatory responses, we have conducted a series of in vitro and in vivo experiments to address the physiological relevance of the moesin-Plasmodium GPI interaction in the context of malaria pathology. We report here that although moesin and Plasmodium GPI interact in vitro, moesin is not critically involved in processes leading to Plasmodium-induced pro-inflammatory immune responses or malaria-associated cerebral pathology.

Generation of glycosylphosphatidylinositol linked chicken IL-17 to generate specific monoclonal antibodies applicable for intracellular cytokine staining.

Interleukin 17 (IL-17) cytokines play a crucial role in host defense and inflammatory diseases. Of the six mammalian IL-17 members five are described in the chicken (gg) genome. A novel method that attached cytokines to the surface of cells via a GPI linker was established to generate two chicken IL-17A and one chicken IL-17F specific mab. Recombinant gg IL-17A and gg IL-17F that showed dimerization in Western blot were used to verify the antibodies specificity. The mab could detect gg IL-17 by intracellular cytokine staining as demonstrated on cells expressing recombinant IL-17. Furthermore IL-17A and lower amounts of IL-17F were detectable in CD4 positive T cells of stimulated splenocytes. In conclusion, we have generated novel tools to analyze chicken IL-17 in more detail and demonstrated that the surface expression of cytokines is a reliable method to generate specific mab applicable for intracellular cytokine staining.

Toward the Development of the Next Generation of a Rapid Diagnostic Test: Synthesis of Glycophosphatidylinositol (GPI) Analogues of Plasmodium falciparum and Immunological Characterization.

A large number of proteins in malaria parasites are anchored using glycophosphatidylinositols (GPIs) with lipid tails. These GPIs are structurally distinct from human GPIs. Plasmodium falciparum GPIs have been considered as potential vaccine candidates because these molecules are involved in inducing inflammatory responses in human hosts, and natural anti-GPI antibody responses have been shown to be associated with protection against severe disease. GPIs can also be considered as targets for rapid diagnostic tests. Because isolation of native GPIs in large quantities is challenging, development of synthetic GPI molecules can facilitate further exploration of GPI molecules for diagnostics. Here, we report synthesis and immunological characterization of a panel of malaria-specific GPI analogues. A total of three GPI analogues were chemically synthesized and conjugated to a carrier protein to immunize and generate antibodies in rabbits. The rabbit immune sera showed reactivity with synthetic GPIs and native GPIs extracted from P. falciparum parasite, as determined by Luminex and ELISA methods.

Understanding the Chemistry and Biology of Glycosylation with Glycan Synthesis.

Glycoscience research has been significantly impeded by the complex compositions of the glycans present in biological molecules and the lack of convenient tools suitable for studying the glycosylation process and its function. Polysaccharides and glycoconjugates are not encoded directly by genes; instead, their biosynthesis relies on the differential expression of carbohydrate enzymes, resulting in heterogeneous mixtures of glycoforms, each with a distinct physiological activity. Access to well-defined structures is required for functional study, and this has been provided by chemical and enzymatic synthesis and by the engineering of glycosylation pathways. This review covers general methods for preparing glycans commonly found in mammalian systems and applying them to the synthesis of therapeutically significant glycoconjugates (glycosaminoglycans, glycoproteins, glycolipids, glycosylphosphatidylinositol-anchored proteins) and the development of carbohydrate-based vaccines.

Reinforcing B16F10/GPI-IL-21 vaccine efficacy against melanoma by injecting mice with shZEB1 plasmid or miR200c agomir.

In this study, we hypothesized that the inhibition of epithelial to mesenchymal transition (EMT) program by knockdown of Zinc-finger E-box binding homeobox 1 (ZEB1) or administration of miR200c agomir would strengthen the B16F10 cells transfected with GPI-anchored IL-21 (B16F10/GPI-IL-21) vaccine efficacy in inhibiting the melanoma metastasis. Our findings from the current study indicated that, when compared with the mice immunized with the B16F10/GPI-IL-21 vaccine alone, the mice immunized with B16F10/GPI-IL-21 vaccine combined with injection of shZEB1 plasmid or miR200c agomir not only meaningfully inhibited EMT of melanoma, reduced the EMT characteristic molecular expression in tumor tissues, but also significantly decreased the Treg cells and TGF-ß1, enhanced the cytotoxicities of NK cells and cytotoxic T lymphocytes and the IFN-γ level. Furthermore, the immunotherapeutic combination resulted in inhibiting the melanoma growth and lung metastasis. Our study demonstrated that using the B16F10/GPI-IL-21 vaccine in combination with the down-regulated ZEB1 or miR200c administration effectively elicited anti-tumor immunity and reduced melanoma metastasis by inhibiting the EMT program in the B16F10 melanoma-bearing mice.

Investigation of the protective properties of glycosylphosphatidylinositol-based vaccine candidates in a Toxoplasma gondii mouse challenge model.

Vaccination against the ubiquitous parasite Toxoplasma gondii would provide the most efficient prevention against toxoplasmosis-related congenital, brain and eye diseases in humans. We investigated the immune response elicited by pathogen-specific glycosylphosphatidylinositol (GPI) glycoconjugates using carbohydrate microarrays in a BALB/c mouse model. We further examined the protective properties of the glycoconjugates in a lethal challenge model using the virulent T. gondii RH strain. Upon immunization, mice raised antibodies that bind to the respective GPIs on carbohydrate microarrays, but were mainly directed against an unspecific GPI epitope including the linker. The observed immune response, though robust, was unable to provide protection in mice when challenged with a lethal dose of viable tachyzoites. We demonstrate that anti-GPI antibodies raised against the here described semi-synthetic glycoconjugates do not confer protective immunity against T. gondii in BALB/c mice.