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Giardia lamblia , one of the most common protozoal infections of the human intestine, is an important worldwide cause of diarrheal disease, malabsorption, malnutrition, delayed cognitive development in children, and protracted postinfectious syndromes. Despite its medical importance, no human vaccine is available against giardiasis. A crude veterinary vaccine has been developed, and experimental vaccines based on expression of multiple variant-specific surface proteins have been reported, but poorly defined vaccine components and excessive antigen variability are problematic for pharmaceutical vaccine production. To expand the repertoire of antigen candidates for vaccines, we reasoned that surface proteins may provide an enriched source of such antigens since key host effectors, such as secretory IgA, can directly bind to such antigens in the intestinal lumen and interfere with epithelial attachment. Here, we have applied a proteomics approach to identify 23 novel surface antigens of G. lamblia that show >90% amino acid sequence identity between the two human-pathogenic genetic assemblages (A and B) of the parasite. Surface localization of a representative subset of these proteins was confirmed by immunostaining. Four selected proteins, uridine phosphorylase-like protein-1, protein 21.1 (GL50803_27925), α1-giardin, and α11-giardin, were subsequently produced in recombinant form and shown to be immunogenic in mice and G. lamblia -infected humans and confer protection against G. lamblia infection upon intranasal immunization in rodent models of giardiasis. These results demonstrate that identification of conserved surface antigens provides a powerful approach for overcoming a key rate-limiting step in the design and construction of an effective vaccine against giardiasis., (Copyright © 2019 American Society for Microbiology.)

Giardia lamblia is present in the intestinal lumen as a binucleate, flagellated trophozoite or a quadranucleate, immotile cyst. Here we used the plant lectin wheat germ agglutinin (WGA), which binds to the disaccharide di-N-acetyl-chitobiose (GlcNAc(2)), which is the truncated Asn-linked glycan (N-glycan) of Giardia, to affinity purify the N-glycomes (glycoproteins with N-glycans) of trophozoites and cysts. Fluorescent WGA bound to the perinuclear membranes, peripheral acidified vesicles, and plasma membranes of trophozoites. In contrast, WGA bound strongly to membranes adjacent to the wall of Giardia cysts and less strongly to the endoplasmic reticulum and acidified vesicles. WGA lectin-affinity chromatography dramatically enriched secreted and membrane proteins of Giardia, including proteases and acid phosphatases that retain their activities. With mass spectroscopy, we identified 91 glycopeptides with N-glycans and 194 trophozoite-secreted and membrane proteins, including 42 unique proteins. The Giardia oligosaccharyltransferase, which contains a single catalytic subunit, preferred N glycosylation sites with Thr to those with Ser in vivo but had no preference for flanking amino acids. The most-abundant glycoproteins in the N-glycome of trophozoites were lysosomal enzymes, folding-associated proteins, and unique transmembrane proteins with Cys-, Leu-, or Gly-rich repeats. We identified 157 secreted and membrane proteins in the Giardia cysts, including 20 unique proteins. Compared to trophozoites, cysts were enriched in Gly-rich repeat transmembrane proteins, cyst wall proteins, and unique membrane proteins but had relatively fewer Leu-rich repeat proteins, folding-associated proteins, and unique secreted proteins. In summary, there are major changes in the Giardia N-glycome with the differentiation from trophozoites to cysts.

The SIMPLE-READ Giardia rapid assay (Medical Chemical Corporation) is a solid-phase qualitative immunochromatographic assay that detects Giardia lamblia in aqueous extracts of human fecal specimens. Testing 106 Giardia-positive and 104 Giardia-negative stool specimens yielded a sensitivity of 97.2% and a specificity of 100% for the SIMPLE-READ Giardia rapid assay.

Eukaryotic translation initiation factor 4E (eIF4E) binds to the m(7)GTP of capped mRNAs and is an essential component of the translational machinery that recruits the 40S small ribosomal subunit. We describe here the identification and characterization of two eIF4E homologues in an ancient protist, Giardia lamblia. Using m(7)GTP-Sepharose affinity column chromatography, a specific binding protein was isolated and identified as Giardia eIF4E2. The other homologue, Giardia eIF4E1, bound only to the m(2,2,7)GpppN structure. Although neither homologue can rescue the function of yeast eIF4E, a knockdown of eIF4E2 mRNA in Giardia by a virus-based antisense ribozyme decreased translation, which was shown to use m(7)GpppN-capped mRNA as a template. Thus, eIF4E2 is likely the cap-binding protein in a translation initiation complex. The same knockdown approach indicated that eIF4E1 is not required for translation in Giardia. Immunofluorescence assays showed wide distribution of both homologues in the cytoplasm. But eIF4E1 was also found concentrated and colocalized with the m(2,2,7)GpppN cap, 16S-like rRNA, and fibrillarin in the nucleolus-like structure in the nucleus. eIF4E1 depletion from Giardia did not affect mRNA splicing, but the protein was bound to Giardia small nuclear RNAs D and H known to have an m(2,2,7)GpppN cap, thus suggesting a novel function not yet observed among other eIF4Es in eukaryotes.

Two hundred twenty-three formalin-preserved stool specimens were evaluated by using ProSpecT Giardia Rapid Assay (membrane bound) (Alexon, Inc., Sunnyvale, Calif.). Enzyme immunoassay (EIA) results were compared with those by conventional microscopic examination. Two hundred four specimens were negative by both methods, and 13 (6.3%) were positive. Five specimens were negative by initial microscopic exam and positive by EIA; three of these specimens were found to be positive upon extensive microscopic reexamination. The remaining two specimens were from patients who previously tested positive and who had recurrent symptoms of or responded to therapy for giardia. Therefore, we consider both cases to be true positives. One specimen exhibited a single cyst by microscopic exam and was negative by EIA Resolved results yielded a relative sensitivity of 95%, a relative specificity of 100%, a positive predictive value of 99.6%, and a negative predictive value of 100%, compared with a sensitivity of 74% for conventional microscopy.