Identification of a putative Crf splice variant and generation of recombinant antibodies for the specific detection of Aspergillus fumigatus

Abstract

Background: Aspergillus fumigatus is a common airborne fungal pathogen for humans. It frequently causes an invasive aspergillosis (IA) in immunocompromised patients with poor prognosis. Potent antifungal drugs are very expensive and cause serious adverse effects. Their correct application requires an early and specific diagnosis of IA, which is still not properly achievable. This work aims to a specific detection of A. fumigatus by immunofluorescence and the generation of recombinant antibodies for the detection of A. fumigatus by ELISA.

Results: The A. fumigatus antigen Crf2 was isolated from a human patient with proven IA. It is a novel variant of a group of surface proteins (Crf1, Asp f9, Asp f16) which belong to the glycosylhydrolase family. Single chain fragment variables (scFvs) were obtained by phage display from a human naive antibody gene library and an immune antibody gene library generated from a macaque immunized with recombinant Crf2. Two different selection strategies were performed and shown to influence the selection of scFvs recognizing the Crf2 antigen in its native conformation. Using these antibodies, Crf2 was localized in growing hyphae of A. fumigatus but not in spores. In addition, the antibodies allowed differentiation between A. fumigatus and related Aspergillus species or Candida albicans by immunofluorescence microscopy. The scFv antibody clones were further characterized for their affinity, the nature of their epitope, their serum stability and their detection limit of Crf2 in human serum.

Conclusion: Crf2 and the corresponding recombinant antibodies offer a novel approach for the early diagnostics of IA caused by A. fumigatus.

Figure 1. Isolation and analysis of crf2.

A Isolation of crf2. PCR products derived from cDNA using primer sets for the amplification of asp f16 (resulting in the amplification of crf2) or asp f9 were separated by 1% agarose gel electrophoresis. B Alignments of the nucleotide sequences of crf1 gene (NCBI: NC_007194.1, CADRE: AFUA_6G08510), mRNA of crf1 (NCBI: AY169706), mRNA of crf2, mRNA of asp f9 (NCBI: AJ223327) and mRNA of asp f16 (NCBI: AF062651). The primer sequences for the amplification of asp f9 and asp f16 are underlined. The asp f16 reverse primer1 binds directly downstream of the crf1 gene stop codon. If the crf1 gene sequence is identical with all mRNA sequences the sequences are red. The crf1 gene sequence is in black and the mRNA sequences are in blue, if differences between the crf1 gene sequence and any mRNA sequence. The leader sequences are marked in green.

Figure 2. Production and purification of Crf2.

IMAC and IEC purified Crf2 was separated on a reducing 12% SDS-PAGE. A Coomassie staining. B the recombinant Crf was detected with mouse anti-his tag (1∶10,000) and goat anti-mouse IgG (Fc specific) AP conjugate (1∶10,000).

Figure 3. Epitope mapping of serum of mice infected with A. fumigatus on Crf1 and Crf2.

A Epitope mapping membrane (15mer oligopeptide, 3 aminoacid overlap) stained with mouse serum (1∶400). The bound mouse antibodies were detected with goat anti-mouse IgG Fc specific AP conjugated (1∶2000). The stained Crf2 specific epitope was marked with a square. B Aligment of Crf1, Crf2 and Asp f9. The recognized epitopes of Crf1 and Crf2 are marked in red (the shorter polypeptide Asp f9 is shown for comparison, Asp f16 is not shown due to a frameshift resulting in a divergent amino acid sequence compared to Crf1, Crf2 and Asp f9).

Figure 4. Schematic overview about of the construction of macaque immune libraries, selection and screening of antibodies.

Figure 5. Immunoblot of the Hyperphage packaged macaque library.

1×10⁹ or 5×10⁹ scFv phage particles or 1×10⁹ Hyperphage particles per lane were separated on a reducing 10% SDS-PAGE, followed by Western blot and detection of wildtype pIII or scFv::pIII fusion using mouse mAb anti-pIII (1∶2000) and goat anti-mouse HRP (1∶5,000).

Figure 6. Fluorescence microscopy using anti-Crf2 scFv-Fc fusion proteins.

A Binding of anti-Crf2 scFv-Fc fusion proteins to A. fumigatus (left panel brightfield, right panel FITC). B Binding of MS112-IIB1 to different Aspergillus strains (A. fumigatus, A. nidulans, A. terreus, A. flavus, A. clavatus) and Candida albicans. The staining was performed using 2 µg/mL scFv-Fc fusion protein and goat anti-human IgG (Fc specific) conjugated with Alexa 488 (1∶500).

Figure 7. Analysis of the antigen binding by titration ELISA to analyse the binding of the anti-Crf2 scFvs to differently immobilized recombinant Crf2.

400 ng biotinylated Crf2 was immobilized on streptavidin microtitre plate to keep the native folding (A) or directly coated onto Maxisorp plates resulting in partial denaturation of Crf2 (B). A dilution series of scFvs were used for Crf2 detection. The bound scFvs were detected with mouse anti-myc (1∶1,000) and goat anti-mouse IgG (Fab specific) HRP conjugate (1∶10,000).

Figure 8. Determination of the affinity of the anti-Crf2 scFvs by surface plasmon resonance.

SPR analyses of scFv MS130i-IIIC3 (A), scFv MS130i-IIIG3 (B).

Figure 9. Analysis of the anti-Crf2 scFv stability by titration ELISA.

Antigen: 400 ng biotinylated Crf2 coated on streptavidin microtitre plates. ScFvs were incubated in PBS (A), human serum (B) and inactivated human serum (C) for 0 to 27 days at 37°C and used for Crf2 detection. The scFvs were detected with mouse anti-myc (1∶1,000) and goat anti-mouse IgG HRP conjugated (1∶10,000).

Figure 10. Detection of recombinant biotinylated Crf2 in human serum using anti-Crf2 scFvs.

400 ng anti-Crf2 scFvs were coated for capturing. Human serum was spiked with a dilution series of recombinant Crf2. The bound Crf2 was detected with 100 µL streptavidin HRP conjugate (1 µg/mL).