anti-GPI Antibodies Research Articles

Synthetic GPI as a candidate anti-toxic vaccine in a model of malaria.

The malaria parasite Plasmodium falciparum infects 5-10% of the world's population and kills two million people annually. Fatalities are thought to result in part from pathological reactions initiated by a malarial toxin. Glycosylphosphatidylinositol (GPI) originating from the parasite has the properties predicted of a toxin; however, a requirement for toxins in general and GPI in particular in malarial pathogenesis and fatality remains unproven. As anti-toxic vaccines can be highly effective public health tools, we sought to determine whether anti-GPI vaccination could prevent pathology and fatalities in the Plasmodium berghei/rodent model of severe malaria. The P. falciparum GPI glycan of the sequence NH(2)-CH(2)-CH(2)-PO(4)-(Man alpha 1-2)6Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcNH(2)alpha 1-6myo-inositol-1,2-cyclic-phosphate was chemically synthesized, conjugated to carriers, and used to immunize mice. Recipients were substantially protected against malarial acidosis, pulmonary oedema, cerebral syndrome and fatality. Anti-GPI antibodies neutralized pro-inflammatory activity by P. falciparum in vitro. Thus, we show that GPI is a significant pro-inflammatory endotoxin of parasitic origin, and that several disease parameters in malarious mice are toxin-dependent. GPI may contribute to pathogenesis and fatalities in humans. Synthetic GPI is therefore a prototype carbohydrate anti-toxic vaccine against malaria.

Rationale for malaria anti-toxin therapy

Glycosylphosphatidylinositols (GPIs) are a distinct class of glycolipids that are present ubiquitously in eukaryotic cells, where they perform a variety of biological processes. GPIs are particularly abundant in protozoa, where they are found as free lipids and attached to proteins. For the asexual blood stages of Plasmodium falciparum, several functionally important parasite proteins, including merozoite surface protein 1 (MSP1), are anchored to the erythrocyte membrane through GPI moieties. Because the enzyme specificity of Plasmodium GPI biosynthesis differs significantly from that of the mammalian host, it has been speculated that parasite GPIs might provide novel targets for antimalarial drugs or vaccines.The pathology of malaria infection has long been considered to be the result of the release of toxins of parasite origin. Tumour necrosis factor-α (TNF- α), which is induced by such malarial toxins, was subsequently identified as a major host mediator of disease. Consequently, its production and that of other pyrogenic cytokines is commonly regarded as a surrogate marker for the initiation of malaria pathological processes. The GPI of P. falciparum has emerged only recently as a candidate toxin, based on its ability to elicit proinflammatory cytokines from macrophages and endothelial cells by activating NFκB transcription factors and on its ability to cause clinical effects in experimental animals that are akin to acute malaria infection (e.g. fever, hypoglycaemia, dyserythropoiesis and vascular damage in the brain). As P. falciparum-infected erythrocytes are sequestered in specific organs, localized elevated toxic responses to the parasite's GPIs can disturb vital physiological functions and cause severe illness and, in the event of cerebral pathology, even death. Antagonists of GPI-mediated signalling and a monoclonal antibody against P. falciparum GPIs can each block the induction of toxicity in vitro, indicating the feasibility of GPI-based immunotherapy.Naik et al.1xGlycosylphosphatidylinositol anchors of Plasmodium falciparum: molecular characterization and naturally elicited antibody response that may provide immunity to malaria pathogenesis. Naik, R.S et al. J. Exp. Med. 2000; 192: 1563–1575Crossref | PubMed | Scopus (161)See all References1 postulate, because P. falciparum GPIs are pathogenicity factors, that semi-immune adults living in regions of malaria endemicity should possess GPI-specific protective immunity. This hypothesis was tested by analysing the anti-GPI antibody response in sera from a large cohort of individuals from western Kenya. Their findings demonstrate for the first time that people in malaria-endemic regions elicit a potent GPI-specific immunoglobulin (Ig) G response, and that this increases in an age-dependent (and possibly exposure-dependent) manner. Thus, although adults and older children have high antibody titres, children susceptible to acute P. falciparum infection either have low levels of short-lived antibodies or none at all. Absence of a persistent anti-GPI antibody response was associated with malaria-specific anaemia and fever. Naik et al.1xGlycosylphosphatidylinositol anchors of Plasmodium falciparum: molecular characterization and naturally elicited antibody response that may provide immunity to malaria pathogenesis. Naik, R.S et al. J. Exp. Med. 2000; 192: 1563–1575Crossref | PubMed | Scopus (161)See all References1 speculate that antibodies to P. falciparum GPIs are involved in protection against clinical infection. This speculation is based on the discovery of a direct correlation between serum anti-GPI IgG and resistance to malaria pathogenesis; a causal link now needs to be established in a suitable experimental model, between the possession of antibodies to parasite GPIs and prevention of pathogenesis. Understanding the factors associated with resistance to clinical infection, so-called anti-disease immunity, might lead to alternative approaches for malaria control. In this regard, GPIs appear to offer a genuine opportunity.

Glycosylphosphatidylinositol anchors of Plasmodium falciparum: molecular characterization and naturally elicited antibody response that may provide immunity to malaria pathogenesis.

Induction of proinflammatory cytokine responses by glycosylphosphatidylinositols (GPIs) of intraerythrocytic Plasmodium falciparum is believed to contribute to malaria pathogenesis. In this study, we purified the GPIs of P. falciparum to homogeneity and determined their structures by biochemical degradations and mass spectrometry. The parasite GPIs differ from those of the host in that they contain palmitic (major) and myristic (minor) acids at C-2 of inositol, predominantly C18:0 and C18:1 at sn-1 and sn-2, respectively, and do not contain additional phosphoethanolamine substitution in their core glycan structures. The purified parasite GPIs can induce tumor necrosis factor alpha release from macrophages. We also report a new finding that adults who have resistance to clinical malaria contain high levels of persistent anti-GPI antibodies, whereas susceptible children lack or have low levels of short-lived antibody response. Individuals who were not exposed to the malaria parasite completely lack anti-GPI antibodies. Absence of a persistent anti-GPI antibody response correlated with malaria-specific anemia and fever, suggesting that anti-GPI antibodies provide protection against clinical malaria. The antibodies are mainly directed against the acylated phosphoinositol portion of GPIs. These results are likely to be valuable in studies aimed at the evaluation of chemically defined structures for toxicity versus immunogenicity with implications for the development of GPI-based therapies or vaccines.

Human glucosamine-6-phosphate isomerase, a homologue of hamster oscillin, does not appear to be involved in Ca2+ release in mammalian oocytes.

Injections of cytosolic preparations from mammalian sperm into oocytes have been shown to trigger calcium [Ca2+]i oscillations and initiate activation of development. Recently, a protein isolated from hamster sperm has been suggested to be involved in the generation of these oscillations and it was named "oscillin." The human homologue of hamster oscillin is glucosamine 6-phosphate isomerase (GPI, EC no. 5.3.1.10), an enzyme so far described to be involved in hexose phosphate metabolism. To assess the role of GPI on Ca2+ signaling, a human recombinant protein was generated in a prokaryotic system and injected into fura-2-dextran-loaded metaphase II (MII) mouse oocytes. Injection of recombinant GPI failed to induce Ca2+ responses in 12/12 injected MII oocytes despite the fact that the recombinant GPI was active as assessed by an enzymatic assay. Injection of buffer (0/6 oocytes) or fructose-6-phosphate, a product of GPI enzymatic reaction (0/5 oocytes), also failed to initiate Ca2+ responses. Conversely, injections of sperm cytosolic factor induced [Ca2+]i oscillations in all 17/17 oocytes. In addition, injection of recombinant GPI or GPI mRNA failed to induce parthenogenetic activation (0/30 oocytes). Immunofluorescence studies using an anti-GPI polyclonal antibody (GK) resulted in localization of GPI to the sperm's equatorial region. Incubation of the GK antibody with sperm extracts failed to block the [Ca2+]i responses induced by these extracts. Moreover, near complete depletion of GPI from sperm fractions by immunoprecipitation did not impair the ability of these fractions to induce [Ca2+]i oscillations. In summary, our results support the role of a sperm cytosolic component(s) in the generation of [Ca2+]i oscillations during mammalian fertilization, although a protein other than GPI/oscillin is likely to be the active calcium releasing factor.

Boosting immune response with a candidate varicella-zoster virus glycoprotein subunit vaccine

A varicella-zoster virus (VZV) seropositive individual was immunized with 100 μg of purified VZV TgpI-511 glycoprotein subunit antigen formulated with monophosphoryl lipid A. Serum samples were obtained during a 40-day period post-immunization (PI) and analysed by immunoprecipitation and virus neutralization tests. The results from immunoprecipitation studies revealed an increase in VZV anti-gpI antibody titer as early as 6 days PI which continued to rise during 40 days PI. In addition, virus neutralization tests showed a 21.0% VZV neutralization 6 days PI with an increase to a 96.7% VZV neutralization 40 days PI. These results suggested that the candidate VZV glycoprotein subunit vaccine (TgpI-511) was capable of boosting the production of neutralizing antibodies in the immunized VZV seropositive human subject.

Immunogenicity in mice of varicella-zoster virus glycoprotein I expressed by a vaccinia virus-varicella-zoster virus recombinant.

Several vaccinia virus (VV)-varicella-zoster virus (VZV) recombinants expressing glycoprotein I (gpI) of VZV were isolated from the Prague strain of VV. One of these, v46, was inoculated intraperitoneally into mice. Groups of mice were bled 4 and 8 weeks later and their sera were examined for anti-VZV and anti-VV antibodies by ELISA. At 4 weeks, all mice inoculated with the three largest virus doses (10(7), 10(6) and 10(5) p.f.u.), and at 8 weeks all mice inoculated with the four highest virus doses (10(7), 10(6), 10(5), and 10(4) p.f.u.), had developed both anti-VV and anti-VZV antibodies. Antibodies were also detected in a high proportion of mice infected with lower doses of virus and in some instances VZV antibodies were present in the absence of VV antibodies. None of the animals inoculated in parallel with either a thymidine kinase-negative mutant of the original VV or diluent alone developed antibody reactive with VZV. The specificity of the reaction was assessed further by Western blotting using anti-gpI monoclonal antibodies as a positive control. Sera from animals immunized with v46 possessed antibody capable of neutralizing extracellular VZV in the presence of complement.

Acute and persistent varicella‐zoster virus infection of human and murine neuroblastoma cell lines

Human and murine neuroblastoma cell lines were infected in vitro with varicella-zoster virus (VZV). Infected human neuroblastoma cells (IMR-32) supported the synthesis of abundant viral antigens as detected by indirect immunoperoxidase labeling using human serum rich in anti-VZV antibodies and did not survive the infection. In situ hybridization (ISH) with VZV-cloned probes revealed a strong hybridization signal in these infected cells. During cultivation, the virus was released in the culture medium, and viral polypeptides were revealed by Western blotting of infected cells, using either a monoclonal anti-gpI antibody or a rabbit antiserum. All these findings indicate that IMR-32 cells support a productive and lytic infection by VZV, whether infected by cell-free virus or by cocultivation with infected cells. Murine neuroblastoma cells (neuro-2A) survived VZV infection and did not produce any infectious virus. No VZV-specific proteins were detected in infected cells either by immunolabeling or by Western blotting. However, viral nucleic acids could be detected by ISH, indicating that mouse neuroblastoma cells displayed a nonproductive, nonlytic infection. Infected neuro-2A cells have been examined by ISH using probes corresponding to immediate early (IE) genes 4, 62, and 63 and late (L) gene 31 encoding gpII. A strong hybridization signal was detected when infected cells were probed with a fragment containing the IE genes 62 and 63. Lower levels of hybridization were detected with the other probes, corresponding to IE or L genes. These systems allow comparative molecular analysis of persistent and acute infection of nerve cells by VZV.

Cross-reactive and unique grass group I antigenic determinants defined by monoclonal antibodies

As part of a study to probe the immunochemical basis for allergenic cross-reactivity among grass pollens, a series of useful reagents has been prepared. The major grass-pollen allergen, designated group I (GpI), was isolated from five grass pollens (meadow fescue, June grass, sweet vernal grass, redtop grass, and perennial ryegrass). The purified GpI antigens were used to immunize individual groups of BALB/c mice. A total of 123 hybridoma-derived anti-GpI monoclonal antibodies (mAbs) was produced. These mAbs were used to evaluate the antigenk relationship among the GpI antigens by means of two types of ELISA. The experiments revealed a high level of epitope diversity and demonstrated a wide range of antibody specificities. A cross-reactivity ELISA was used to identify and compare antigenic determinants on the GpI molecules, and it was possible to define mAbs with specificities unique for the immunizing allergen and other mAbs that cross-reacted with one or more other members of the test panel of allergens. These murine mAbs reflect the range of specificities present in sera from grass-allergic individuals.

Immunoreactivity of the two common allozymes of murine glucosephosphate isomerase.

There are two common electrophoretic variants (allozymes) of murine glucosephosphate isomerase (GPI). In order to develop immunocytochemical procedures that are allozyme-specific, the two variants (GPI-1A and GPI-1B) were purified from skeletal muscle of several mouse strains and used as antigens for the elicitation of antibodies. The allozymes were purified to a specific activity of 800 units/mg by substrate elution from cellulose phosphate. When the purified allozymes were presented as antigens to goats, rabbits, and mice (both syngeneically and allogeneically), the goats and rabbits produced high titers of anti-GPI antibody, but no humoral antibody was detected in the mice, as determined by radioimmunoassays. Antisera specific for the GPI-1B variant were enriched by absorbing selected sera with GPI-1A conjugated to Sepharose 4B. No antisera specific for GPI-1A were detected following the immunizations. The specificity of the anti-GPI-1B antisera provides a unique opportunity for the development of immunocytochemical procedures for studying the distribution fo this allozyme in chimeric mouse tissues.

Immunofluorescence and histochemical localization of glucosephosphate isomerase in neural tissues.

The distribution of glucosephosphate isomerase (GPI, D-glucose-6-phosphate ketol isomerase) in mouse nervous tissue has been determined at the light microscopic level by immunofluorescence and histochemical procedures. The fluorescence procedure, which utilizes anti-GPI antibodies, detected lower levels of GPI than the histochemical procedure, which relies upon the catalytic activity of the enzyme. The distribution of GPI in nervous tissue is very similar to that of hexokinase. High levels of GPI were found in the Purkinje cells, the molecular layer, and the glomeruli of the granular layer in the cerebellar cortex; the pontine nuclei and the inferior olivary nuclei of the pons and medulla; the neurons of the thalamus and hypothalamus; the pyramidal cells, the dentate nuclei, and Ammons' horn of the cerebral cortex; the ventral horn cells of the spinal cord; and ventricular cells, choroid plexus cells, and the leptomeninges. The neuropil throughout the central nervous system (CNS) stained uniformly with moderately high levels of GPI. No GPI was observed in the myelin sheaths of the CNS.