Production and characterization of monoclonal antibodies against Encephalitozoon intestinalis and Encephalitozoon sp. spores and their developmental stages

Abstract

Background: Microsporidia are intracellular obligate parasites traditionally associated with immunosuppressed patients; their detection in immunocompetent patients has increased, highlighting their possible importance as emerging pathogens. Detection of spores in stools, urine, body fluids and tissues is difficult and immunological techniques such as immunofluorescence have proved to be a useful and reliable tool in the diagnosis of human microsporidiosis. For this reason, we have produced and characterized monoclonal antibodies (MAbs) specific for Encephalitozoon intestinalis (the second most frequent microsporidian infecting humans), and other Encephalitozoon species, that can be used in different diagnostic techniques.

Results: Seven MAbs were selected in accordance with their optical density (OD). Four (4C4, 2C2, 2E5 and 2H2) were isotype IgG2a; two (3A5 and 3C9) isotype IgG3, and one Mab, 1D7, IgM isotype. The selected monoclonal antibody-secreting hybridomas were characterized by indirect immunofluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA), Western blot, immunoelectron microscopy (Immunogold) and in vitro cultures. The study by IFAT showed different behavior depending on the MAbs studied. The MAbs 4C4, 2C2, 2E5 and 2H2 showed reactivity against epitopes in the wall of the spore (exospore and endospore) epitopes located in Encephalitozoon sp. spores, whereas the MAbs 3A5, 1D7 and 3C9 showed reactivity against internal epitopes (cytoplasmic contents or sporoplasm) of E. intestinalis spores. All MAbs recognized the developing parasites in the in vitro cultures of E. intestinalis. Additionally, 59 formalin-fixed stool samples that had been previously analyzed were screened, with 26 (44%) presenting microsporidian spores (18 samples with E. intestinalis and 8 samples with Enterocytozoon bieneusi). Detection of microsporidian spores by microscopy was performed using Calcofluor stain, Modified Trichrome, Quick-Hot Gram Chromotrope, as well as IFAT using MAbs 4C4, 2C2, 2E5 and 2H2. The 4 MAbs tested clearly recognized the larger spores corresponding to E. intestinalis, but showed no reactivity with Enterocytozoon bieneusi spores. The mass spectrometry and proteomic study revealed that the Mabs 4C4, 2C2, 2E5 and 2H2 recognized the Spore Wall Protein 1 (SWP1) as the antigenic target.

Conclusions: The IFAT-positive MAbs exhibited excellent reactivity against spores and developmental stages, permitting their use in human and animal diagnosis. The epitopes recognized (exospore, endospore and cytoplasmic contents) by the different MAbs developed need further study, and may reveal potential targets for vaccine development, immunotherapy and chemotherapy.

Keywords: Developmental stages; Diagnosis; Encephalitozoon intestinalis; Encephalitozoon sp.; Monoclonal antibodies; Spores.

Fig. 1

Western blot of SDS-PAGE (12%) separated proteins of E. intestinalis. Undiluted monoclonal antibodies: Pattern 1 (MAbs 4C4, 2C2 and 2H2); Pattern 2 (MAb 2E5); Pattern 3 (MAbs 3C9 and 1D7); Pattern 4 (MAb 3A5)

Fig. 2

Immunogold electron micrographs of Encephalitozoon intestinalis spores after incubation with MAbs. Undiluted supernatant were used: 2C2 (a), 3C9 (b), 2E5 (c) and 4C4 (d). Scale-bars: a, 0.3 μm; b, 0.25 μm; c, 0.5 μm; d, 0.2 μm

Fig. 3

Immunorecognized in culture cell (Vero-E6) of E. intestinalis after incubation with MAb 2E5 (undiluted supernatant). a 24 hpi. b 72 hpi. Abbreviations: N, nucleus; Pv, parasitophorous vacuole; Sd, stages of development; hpi, hours post-infection

Fig. 4

Formalin-fixed fecal samples from patients with microsporidia. a Fecal smear stained by the Modified Trichrome technique. b Encephalitozoon intestinalis spores detected by IFAT with the MAb 2E5. c and d are the same field: c Fecal smear stained by the Calcofluor technique. d Spores detected by IFAT with MAb 2C2. Scale-bars: 4 μm

Fig. 5

Soluble antigen profiles of E. cuniculi. a Soluble antigen in NuPage® gel stained with Coomasie Blue. b Immunoreactivity profile of MAb 2C2 (dilution 1:20, iBlot™ Dry Blotting System, Invitrogen™)