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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

Structural alignment of the capsid protein VP1. The resolved VP1 structure of 6 enterovirus types (CV-B1, CV-B3, CV-A24, CV-A21, E-1, E-7) was aligned in order to identify highly conserved regions.(a) Soluble VP1: the 5D-8.1 epitope (yellow) and the 9D5 epitope (green) are indicated. (b) VP1 assembled into an enterovirus capsomere. (c) Localization of VP1 within the enterovirus capsid.
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f3: Structural alignment of the capsid protein VP1. The resolved VP1 structure of 6 enterovirus types (CV-B1, CV-B3, CV-A24, CV-A21, E-1, E-7) was aligned in order to identify highly conserved regions.(a) Soluble VP1: the 5D-8.1 epitope (yellow) and the 9D5 epitope (green) are indicated. (b) VP1 assembled into an enterovirus capsomere. (c) Localization of VP1 within the enterovirus capsid.

Mentions: For each viral capsid organization level, the Solvent-Accessible Surface Area (SASA) was calculated in order to estimate the degree-of-burial of antibody epitopes within the protein. The resolved structures of six reference enterovirus strains were obtained from the RCSB database and a 1.4 Å sphere probe was used to represent a water molecule. The exposed surface area was first calculated, and then normalized with the maximum allowed solvent-accessible area30. Normalized SASA values take the form of Relative Solvent Accessibility (RSA), a quantity which varies between 0 (for completely buried residues) and 100 (for maximally exposed residues). Results are summarized in Fig. 2. The alignment of the VP1 regions recognized by the two MAbs is shown in Fig. 3a. The target residues are mainly accessible in the monomeric form for the two epitopes (N-terminal 5D-8.1, yellow; C- terminal 9D5, green). The exposed residues are highly conserved among different EV types, evidencing their importance in the capsid assembly process. Localization of the VP1 protein within the capsomere (Fig. 3b) shows that the 5D-8.1 epitope is located in a domain where exposed residues are stabilized by a beta sheet structure. Figure 3c shows the two epitopes in the VP1 protein assembled into the capsid.


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

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

Structural alignment of the capsid protein VP1. The resolved VP1 structure of 6 enterovirus types (CV-B1, CV-B3, CV-A24, CV-A21, E-1, E-7) was aligned in order to identify highly conserved regions.(a) Soluble VP1: the 5D-8.1 epitope (yellow) and the 9D5 epitope (green) are indicated. (b) VP1 assembled into an enterovirus capsomere. (c) Localization of VP1 within the enterovirus capsid.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Structural alignment of the capsid protein VP1. The resolved VP1 structure of 6 enterovirus types (CV-B1, CV-B3, CV-A24, CV-A21, E-1, E-7) was aligned in order to identify highly conserved regions.(a) Soluble VP1: the 5D-8.1 epitope (yellow) and the 9D5 epitope (green) are indicated. (b) VP1 assembled into an enterovirus capsomere. (c) Localization of VP1 within the enterovirus capsid.
Mentions: For each viral capsid organization level, the Solvent-Accessible Surface Area (SASA) was calculated in order to estimate the degree-of-burial of antibody epitopes within the protein. The resolved structures of six reference enterovirus strains were obtained from the RCSB database and a 1.4 Å sphere probe was used to represent a water molecule. The exposed surface area was first calculated, and then normalized with the maximum allowed solvent-accessible area30. Normalized SASA values take the form of Relative Solvent Accessibility (RSA), a quantity which varies between 0 (for completely buried residues) and 100 (for maximally exposed residues). Results are summarized in Fig. 2. The alignment of the VP1 regions recognized by the two MAbs is shown in Fig. 3a. The target residues are mainly accessible in the monomeric form for the two epitopes (N-terminal 5D-8.1, yellow; C- terminal 9D5, green). The exposed residues are highly conserved among different EV types, evidencing their importance in the capsid assembly process. Localization of the VP1 protein within the capsomere (Fig. 3b) shows that the 5D-8.1 epitope is located in a domain where exposed residues are stabilized by a beta sheet structure. Figure 3c shows the two epitopes in the VP1 protein assembled into the capsid.

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