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. 2018 Mar 26;56(4):e01566-17.
doi: 10.1128/JCM.01566-17. Print 2018 Apr.

Development and Evaluation of a Broad Bead-Based Multiplex Immunoassay To Measure IgG Seroreactivity against Human Polyomaviruses

Sergio Kamminga  1   2 , Els van der Meijden  3 , Herman F Wunderink  3 , Antoine Touzé  4 , Hans L Zaaijer #  2 , Mariet C W Feltkamp #  1
Affiliations

Development and Evaluation of a Broad Bead-Based Multiplex Immunoassay To Measure IgG Seroreactivity against Human Polyomaviruses

Sergio Kamminga et al. J Clin Microbiol. .

Abstract

The family of polyomaviruses, which cause severe disease in immunocompromised hosts, has expanded substantially in recent years. To accommodate measurement of IgG seroresponses against all currently known human polyomaviruses (HPyVs), including the Lyon IARC polyomavirus (LIPyV), we extended our custom multiplex bead-based HPyV immunoassay and evaluated the performance of this pan-HPyV immunoassay. The VP1 proteins of 15 HPyVs belonging to 13 Polyomavirus species were expressed as recombinant glutathione S-transferase (GST) fusion proteins and coupled to fluorescent Luminex beads. Sera from healthy blood donors and immunocompromised kidney transplant recipients were used to analyze seroreactivity against the different HPyVs. For BK polyomavirus (BKPyV), the GST-VP1 fusion protein-directed seroresponses were compared to those obtained against BKPyV VP1 virus-like particles (VLP). Seroreactivity against most HPyVs was common and generally high in both test populations. Low seroreactivity against HPyV9, HPyV12, New Jersey PyV, and LIPyV was observed. The assay was reproducible (Pearson's r2 > 0.84, P < 0.001) and specific. Weak but consistent cross-reactivity between the related viruses HPyV6 and HPyV7 was observed. The seroresponses measured by the GST-VP1-based immunoassay and a VP1 VLP-based enzyme-linked immunosorbent assay were highly correlated (Spearman's ρ = 0.823, P < 0.001). The bead-based pan-HPyV multiplex immunoassay is a reliable tool to determine HPyV-specific seroresponses with high reproducibility and specificity and is suitable for use in seroepidemiological studies.

Keywords: immunoassay; immunology; polyomavirus; seroepidemiology.

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Figures

FIG 1
FIG 1
Expression and coupling of HPyV VP1 to polystyrene beads. (A) Coomassie-stained SDS-PAGE gel showing glutathione-purified GST-VP1 bacterial lysates of JCPyV, KIPyV, WUPyV, MWPyV, STLPyV, HPyV10, NJPyV, HPyV12, and LIPyV. Numbers in parentheses display the molecular masses (in kilodaltons) of the GST-VP1 fusion proteins. The molecular masses (in kilodaltons) of the PageRuler prestained protein ladder (Thermo Fisher Scientific, Waltham, MA, USA) are indicated on the left. The lane for LIPyV was added at a later date. (B) Purification and coupling of GST-VP1.tag fusion proteins of JCPyV, KIPyV, WUPyV, MWPyV, HPyV10, STLPyV, HPyV12, NJPyV, and LIPyV to glutathione-casein cross-linked beads. GST-VP1-containing crude bacterial extracts were serially diluted (1 to 0.25 mg/ml). GST-VP1.tag coupling, detected by using anti-tag antibodies followed by anti-mouse immunoglobulin-phycoerythrin antibodies, is depicted as the median fluorescence intensity (MFI), measured in a Bio-Plex 100 analyzer.
FIG 2
FIG 2
Seroresponses against each GST-HPyV VP1 antigen measured in the multiplex immunoassay. Seroreactivity was measured in a cohort of healthy blood donors (HBD; n = 87) (A) and a cohort of kidney transplant recipients (KTR; n = 65) (B). The results are depicted as the median fluorescence intensity (MFI), measured in a Bio-Plex 100 analyzer. Each circle represents one serum sample.
FIG 3
FIG 3
Summary of observed cross-reactivity between individual HPyV VP1 antigens. The data in the upper right show the percent VP1 sequence similarity based on a pairwise alignment obtained using Geneious software (version 10.0.9) with default ClustalW settings. The data in the lower left show Spearman correlation coefficients (ρ) calculated on the basis of the seroresponses measured against VP1 of the HPyV types tested in the HBD cohort. nd, Spearman correlation coefficients were not determined for these HPyVs.
FIG 4
FIG 4
Comparison of seroreactivity between MWPyV and HPyV10, both of which belong to Polyomavirus species 10. MWPyV and HPyV10 seroreactivities were measured in a cohort of healthy blood donors (HBD). Results are depicted as the median fluorescence intensity (MFI), measured in a Bio-Plex 100 analyzer, for MWPyV on the x axis and for HPyV10 on the y axis. The Spearman correlation coefficient is depicted. Each circle represents one serum sample, and the line represents the results of linear regression analyses.
FIG 5
FIG 5
Analysis of cross-reactivity of polyomavirus seroresponses by VP1-specific competition. Titrated serum samples were preincubated with crude bacterial extract containing GST alone (black), with GST-VP1 of the autologous HPyV (orange), or with the nontarget heterologous HPyVs (gray). Blue lines indicate competition by VP1 other than the target analyte. Results are depicted as median fluorescence intensity (MFI), measured in a Bio-Plex 100 analyzer and shown for the seroresponses measured for BKPyV (A), JCPyV (B), KIPyV (C), WUPyV (D), MCPyV (E), HPyV6 (F), HPyV7 (G), TSPyV (H), HPyV9 (I); MWPyV (J), HPyV10 (K), and STLPyV (L).
FIG 6
FIG 6
Comparison between the GST-VP1 bead-based assay and the VLP-based ELISA for BKPyV. The seroreactivities of kidney transplantation donors (n = 396) were measured by both the bead-based GST-VP1 immunoassay and the VP1 VLP ELISA for BKPyV. Each circle represents one serum sample, and the black line indicates the correlation between the bead-based measurement (MFI) and the ELISA (OD). (Adapted with permission from reference .)
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