Compensating for cross-reactions using avidity and computation in a suspension multiplex immunoassay for serotyping of Zika versus other flavivirus infections

Abstract

The recent spread of Zika virus (ZIKV) in the Americas and Asia necessitates an increased preparedness for improved maternal and perinatal health and blood safety. However, serological cross-reactions, especially to Dengue virus (DENV), complicate ZIKV antibody serodiagnosis. A novel "pan-Flavi" suspension multiplex immunoassay (PFSMIA) using 25 antigens, whole virus (WV), non-structural protein 1 (NS1), and envelope (E) proteins, from 7 zoonotic flaviviruses for specific detection of ZIKV and DENV IgM and IgG was developed. Patterns of antibody cross-reactivity, avidity, and kinetics were established in 104 sera from returning travelers with known ZIKV and DENV infections. PFSMIA gave IgM- and IgG-sensitivities for both viruses of 96-100%, compared to an immunofluorescence assay. Main IgM cross-reactions were to NS1, for IgG to the E and WV antigens. Infecting virus yielded reactivity to several antigens of the homologous virus, while cross-reactions tended to occur only to a single antigen from heterologous virus(es). A specificity-enhancing computer procedure took into account antibody isotype, number of antibody-reactive antigens per virus, avidity, average degree of cross-reactivity to heterologous flavivirus antigens, and reactivity changes in serial sera. It classified all 50 cases correctly. Applied to sera from 200 pregnant women and 173 blood donors from Sweden, one blood donor was found ZIKV NS1 IgM positive, and another as ZIKV NS1 IgG positive. These samples did not react with other ZIKV antigens and were thereby judged as false-positives. PFSMIA provided sensitive and specific ZIKV and DENV serology, warranting high-throughput serological surveillance and a minimized need for laborious and expensive virus neutralization assays.

Keywords: Dengue virus; Flavivirus; Pathogen surveillance; Serological cross-reaction; Suspension multiplex immunoassay; Zika virus.

Fig. 1

Evolution of anti-flavivirus antibodies in a ZVD case (Z3) with few cross-reactions. Original data after cutoff subtraction (left panels, corresponding to step 2 of Table 1) and processed final scores (“Data reduction,” with a score cutoff of 1, right panels, corresponding to step 6 of Table 1), for IgM and IgG, with or without urea treatment, are shown. Results from sera taken 10–233 days post first symptom (dpfs) are shown). Probable cross-reactions are shown as arrows. MFI Median Fluorescence Intensity. X-axis antigens are whole virus (“WV”), NS1 or recombinant envelope (“env” or “E”), CHIKV antigens are recombinant E1 protein, wild type (“wt”) and mutated (“m”) from two different manufacturers. DENV antigens are WV and NS1 for each serotype, and summed. ZIKV antigens included recombinant E from two different manufacturers. WNV antigens included WV from two manufacturers (See “Materials and methods”)

Fig. 2

Evolution of anti-flavivirus antibodies in a ZVD case (Z1) with a more complex serological pattern. Original data after cutoff subtraction (left column) and processed final scores (right column), for IgM and IgG, with or without urea treatment, respectively, are shown. For further explanation, see legend of Fig. 1

Fig. 3

Antibody evolution in a multiply sampled DF case (D51). Avidity index (AI) was calculated for DENV NS1 IgG. See legend of Fig. 1 for further explanations

Fig. 4

Temporal evolution of homo- and heterologous flavivirus antibodies in 13 ZVD (a) and 37 DF (b) cases, in sera where the days post first symptom (dpfs; shown in ¹⁰log form) were known. Lines depicting tendencies for IgG and IgM evolution with (GU, MU, dotted lines) and without (G, M, whole lines) urea treatment are shown. For abbreviations, see legend of Fig. 1

Fig. 5

Extent and frequency of probable cross-reactions, represented as the average MFI ratio between heterologous, recipient, flavivirus antigen (”To”), and donor flavivirus (”From”) signal, for DENV WV + NS1 and for ZIKV NS1, respectively. Only cross-reactions of more than 100 MFI were included. Error bars denote standard error of mean. See legend of Fig. 1 for further explanations. Results from patients with known YFV and TBEV vaccinations were excluded

Fig. 6

Avidity indices (AI; ratio of MFI after and before urea, Y-axis) of the antibody reactions of selected antigens with sera from patients with DF (n = 61) and ZVD (n = 43). Upper frames IgM, lower frames IgG. Each frame shows the AIs for a DENV, ZIKV, or TBEV antigen, either WV (WV) or NS1. AIs were not calculated if the reactivity of urea untreated bead was less than 300 MFI. Sera with such results are shown as −0.1 on the Y-axis. Only results with selected antigens are shown

Fig. 7

Blood donor sera which reacted strongly with ZIKV NS1. a A blood donor serum reactive in IgG. b A blood donor serum reactive in IgM. For abbreviations, see the legend of Fig. 1