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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 (GPI 1 ) for the detection of GPI 1 -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 GPI 1 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 GPI 1 -based test is in excellent agreement with the results for the commercial ELISA, as the ROC analysis of the GPI 1 test shows 97% specificity and 98% sensitivity for the assay. GPI 1 was a more reliable predictor for a parasite-specific IgM response compared to SAG1, indicating that a bead-based multiplex assay using GPI 1 in combination with SAG1 may strengthen Toxoplasma gondii serology, in particular in seroepidemiological studies.

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.

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.

A polypeptide with N-acetylgalactosaminylphosphotransferase (GalNAcPTase) activity is present at the cell surface in stable association with adhesion molecules of the cadherin family. Antibodies directed against the GalNAcPTase inhibit homophilic cadherin-mediated adhesion and cadherin-mediated neurite outgrowth. We have determined that the GalNAcPTase is anchored at the cell surface by a glycophosphatidylinositol linkage. Treatment of intact cells with phosphatidylinositol-specific phospholipase C (PI-PLC) releases active GalNAcPTase and abolishes the ability of anti-GalNAcPTAse antibodies to modulate cadherin-mediated adhesion or neurite outgrowth. Furthermore, GalNAcPTase released from cells by PI-PLC is recognized by anti-CRD antiserum and is radiolabeled in cells incubated with [14C]-ethanolamine.

The kinetics of binding of a glycolipid-anchored protein (the promastigote surface protease, PSP) to planar lecithin bilayers is studied by an integrated optics technique, in which the bilayer membrane is supported on an optical wave guide and the phase velocities of guided light modes in the wave guide are measured. From these velocities, the optical parameters of the membrane and PSP layers deposited on the waveguide are determined, yielding in particular the mass of PSP bound to the membrane, which is followed in real time. From a comparison of the binding rates of PSP and PSP from which the lipid moiety has been removed, it is shown that the lipid moiety plays a key role in anchoring the protein to the membrane. Specific and nonspecific binding of antibodies to membrane-anchored PSP is also investigated. As little as a fifth of a monolayer of PSP is sufficient to suppress the appreciable nonspecific binding of antibodies to the membrane.