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Properties of Two Enterovirus Antibodies that are Utilized in Diabetes Research.

Maccari G, Genoni A, Sansonno S, Toniolo A - Sci Rep (2016)

Bottom Line: Immunohistochemical studies with the anti-EV antibody 5D-8.1 have shown that the EV capsid antigen is present in pancreatic islet cells of diabetic subjects.When it was noticed that 5D-8.1 may cross-react with human proteins, doubt was casted on the significance of the above histopathologic findings.However, their epitopes may align with a few human proteins at low expected values.

View Article: PubMed Central - PubMed

Affiliation: Center for Nanotechnology and Innovation, Italian Institute of Technology, Pisa, Italy.

ABSTRACT
Human enteroviruses (EVs) comprise >100 different types. Research suggests a non-chance association between EV infections and type 1 diabetes. Immunohistochemical studies with the anti-EV antibody 5D-8.1 have shown that the EV capsid antigen is present in pancreatic islet cells of diabetic subjects. When it was noticed that 5D-8.1 may cross-react with human proteins, doubt was casted on the significance of the above histopathologic findings. To address this issue, properties of EV antibodies 5D-8.1 and 9D5 have been investigated using peptide microarrays, peptide substitution scanning, immunofluorescence of EV-infected cells, EV neutralization assays, bioinformatics analysis. Evidence indicates that the two antibodies bind to distinct non-neutralizing linear epitopes in VP1 and are specific for a vast spectrum of EV types (not for other human viruses). However, their epitopes may align with a few human proteins at low expected values. When tested by immunofluorescence, high concentrations of 5D-8.1 yelded faint cytoplasmic staining in uninfected cells. At reduced concentrations, both antibodies produced dotted staining only in the cytoplasm of infected cells and recognized both acute and persistent EV infection. Thus, the two monoclonals represent distinct and independent probes for hunting EVs in tissues of patients with diabetes or other endocrine conditions.

No MeSH data available.


Related in: MedlinePlus

Spot patterns produced by MAbs 5D-8.1 (a) and 9D5 (b) with overlapping peptides of the VP1 protein of the following viruses: CV-B1, CV-B4, E-30, CV-A1. Microarray signals were converted to a matrix representation: 5D8.1 (c), and 9D5 (d). Background noise was reduced by multiplying the signal with the moving average of the intensity plot.
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f1: Spot patterns produced by MAbs 5D-8.1 (a) and 9D5 (b) with overlapping peptides of the VP1 protein of the following viruses: CV-B1, CV-B4, E-30, CV-A1. Microarray signals were converted to a matrix representation: 5D8.1 (c), and 9D5 (d). Background noise was reduced by multiplying the signal with the moving average of the intensity plot.

Mentions: Secondary goat anti-mouse IgG Ab did not show background interactions with antigen-derived peptides. As shown in Fig. 1a,c, MAb 5D-8.1 gave defined spots for the VP1 sequence of CV-B1, CV-B4, E-30, and reduced reactivity with CV-A1. The peptide scan indicated IPALTAVETGHT as the consensus sequence containing the epitope of MAb 5D-8.1. Comparable signals were produced by MAb 9D5 (Fig. 1b,d) and attributed to the consensus motif SIGNAYSMFYDG. Thus, relative to the VP1 sequence of CV-B4 (GI: 61031)29, the sequence containing the epitope of 5D-8.1 was close to the N-terminus at AA residues 28–39, whereas that of 5D9 was located towards the C-terminus at residues 187–198. Substitution scan of peptide IPALTAVETGHT against MAb 5D-8.1 allowed to delimit the antibody binding site to a conserved core motif 4-IPALTAAET-12. AA positions 4I, 7L and 8T showed the highest degree of sequence conservation with a nearly complete loss in binding of MAb 5D-8.1 upon exchange by other amino acids. AA positions 5P and 11E were well-conserved, exchange by Q and by H, respectively, caused a 60–80% loss in antibody binding. Compared to this, AA positions 6A and 10A exhibited a slightly higher susceptibility for substitution by selected amino acids with a maximal decrease of 50% of spot intensities. 9A and 12T showed the highest tolerance for exchange by other amino acids. Replacement by F (9A) and A (12T) was accepted without loss in antibody binding.


Properties of Two Enterovirus Antibodies that are Utilized in Diabetes Research.

Maccari G, Genoni A, Sansonno S, Toniolo A - Sci Rep (2016)

Spot patterns produced by MAbs 5D-8.1 (a) and 9D5 (b) with overlapping peptides of the VP1 protein of the following viruses: CV-B1, CV-B4, E-30, CV-A1. Microarray signals were converted to a matrix representation: 5D8.1 (c), and 9D5 (d). Background noise was reduced by multiplying the signal with the moving average of the intensity plot.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835795&req=5

f1: Spot patterns produced by MAbs 5D-8.1 (a) and 9D5 (b) with overlapping peptides of the VP1 protein of the following viruses: CV-B1, CV-B4, E-30, CV-A1. Microarray signals were converted to a matrix representation: 5D8.1 (c), and 9D5 (d). Background noise was reduced by multiplying the signal with the moving average of the intensity plot.
Mentions: Secondary goat anti-mouse IgG Ab did not show background interactions with antigen-derived peptides. As shown in Fig. 1a,c, MAb 5D-8.1 gave defined spots for the VP1 sequence of CV-B1, CV-B4, E-30, and reduced reactivity with CV-A1. The peptide scan indicated IPALTAVETGHT as the consensus sequence containing the epitope of MAb 5D-8.1. Comparable signals were produced by MAb 9D5 (Fig. 1b,d) and attributed to the consensus motif SIGNAYSMFYDG. Thus, relative to the VP1 sequence of CV-B4 (GI: 61031)29, the sequence containing the epitope of 5D-8.1 was close to the N-terminus at AA residues 28–39, whereas that of 5D9 was located towards the C-terminus at residues 187–198. Substitution scan of peptide IPALTAVETGHT against MAb 5D-8.1 allowed to delimit the antibody binding site to a conserved core motif 4-IPALTAAET-12. AA positions 4I, 7L and 8T showed the highest degree of sequence conservation with a nearly complete loss in binding of MAb 5D-8.1 upon exchange by other amino acids. AA positions 5P and 11E were well-conserved, exchange by Q and by H, respectively, caused a 60–80% loss in antibody binding. Compared to this, AA positions 6A and 10A exhibited a slightly higher susceptibility for substitution by selected amino acids with a maximal decrease of 50% of spot intensities. 9A and 12T showed the highest tolerance for exchange by other amino acids. Replacement by F (9A) and A (12T) was accepted without loss in antibody binding.

Bottom Line: Immunohistochemical studies with the anti-EV antibody 5D-8.1 have shown that the EV capsid antigen is present in pancreatic islet cells of diabetic subjects.When it was noticed that 5D-8.1 may cross-react with human proteins, doubt was casted on the significance of the above histopathologic findings.However, their epitopes may align with a few human proteins at low expected values.

View Article: PubMed Central - PubMed

Affiliation: Center for Nanotechnology and Innovation, Italian Institute of Technology, Pisa, Italy.

ABSTRACT
Human enteroviruses (EVs) comprise >100 different types. Research suggests a non-chance association between EV infections and type 1 diabetes. Immunohistochemical studies with the anti-EV antibody 5D-8.1 have shown that the EV capsid antigen is present in pancreatic islet cells of diabetic subjects. When it was noticed that 5D-8.1 may cross-react with human proteins, doubt was casted on the significance of the above histopathologic findings. To address this issue, properties of EV antibodies 5D-8.1 and 9D5 have been investigated using peptide microarrays, peptide substitution scanning, immunofluorescence of EV-infected cells, EV neutralization assays, bioinformatics analysis. Evidence indicates that the two antibodies bind to distinct non-neutralizing linear epitopes in VP1 and are specific for a vast spectrum of EV types (not for other human viruses). However, their epitopes may align with a few human proteins at low expected values. When tested by immunofluorescence, high concentrations of 5D-8.1 yelded faint cytoplasmic staining in uninfected cells. At reduced concentrations, both antibodies produced dotted staining only in the cytoplasm of infected cells and recognized both acute and persistent EV infection. Thus, the two monoclonals represent distinct and independent probes for hunting EVs in tissues of patients with diabetes or other endocrine conditions.

No MeSH data available.


Related in: MedlinePlus