Examination of seroprevalence of coronavirus HKU1 infection with S protein-based ELISA and neutralization assay against viral spike pseudotyped virus

Abstract

Background: Human coronavirus HKU1 (HCoV-HKU1) is a recently identified coronavirus with a global distribution and known to cause mainly respiratory infections.

Objectives: To investigate the seroepidemiology of HKU1 infections in our local population.

Study design: An ELISA-based IgG antibody detection assay using recombinant HCoV-HKU1 nucleocapsid and spike (S) proteins (genotype A) were developed for the diagnosis of CoV-HKU1 infections, Additionally, a neutralization antibody assay using the HCoV-HKU1 pseudotyped virus was developed to detect the presence of neutralizing antibodies in serum with antibody positivity in an S protein-based ELISA.

Results: Results of the recombinant S protein-based ELISA were validated with Western blot, immunofluorescence analysis and flow cytometry. The coupled results demonstrated good correlation with Spearmen's coefficient of 0.94. Seroepidemiological study in a local hospital-based setting using this newly developed ELISA showed steadily increasing HCoV-HKU1 seroprevalence in childhood and early adulthood, from 0% in the age group of <10 years old to a plateau of 21.6% in the age group of 31-40 years old.

Conclusions: Our study demonstrated the usefulness of the S-based ELISA which could be applied to future epidemiological studies of HCoV-HKU1 in other localities.

Fig. 1

Determination of cutoff baseline from 297 patients. Two ELISA-based assays against HKU1 recombinant proteins nucleocapsid (N) and spike (S) were compared. Mean OD ±3S.D. is 0.534 and 0.495 for N and S-based ELISA. Serum samples with OD above the baseline values were confirmed by Western blot.

Fig. 2

Western blot analysis can rectify non-specificities of ELISA against recombinant HCoV-HKU1 nucleocapsid (N). Shown are 21 N-based ELISA seropositive samples (lanes 2–22). HKU1-index patient (lane 1, serum sample S0); ELISA positive in both N and S (lanes 2–16, serum samples S1–S15); ELISA positive in N but negative for S (lanes 17–22) and ELISA negative in both N and S (lanes 12–13), chemiluminescent signals were detected using ECL substrate.

Fig. 3

(A) Detection of neutralizing antibodies in serum samples with HCoV-HKU1 infection which inhibit the infection of A549 cells by blocking entry of pHIV-GFP/HKU1 pseudotyped virus. Neutralizing antibodies targeted against pseudovirus bearing HKU1-spike do not neutralize the infection by pseudotyped virus bearing VSV-G protein. (B–D) Specificities of HKU1 pseudotyped viruses were shown against other convalescent patient serum from other SARS and other non-SARS human coronaviruses, OC-43 and 229E. (E). Western blot showed no cross-reactivity between HKU1-S antigen with other coronavirus infected patient serum tested in (B). Lane 1: HKU1 index patient serum, lanes 2–6 HCoV-OC43 patient sera, lanes 7–10 HCoV 229E patient serum and lanes 11–15 SARS patient sera.

Fig. 4

Detection of antibodies against native HCoV-HKU1 S expressed in BHK-21 cells by flow cytometry. Sera A and B (C1–2) are S-based-ELISA negative samples. Serum C (S0) was taken from a patient who had recovered from HKU1 infection. Sera D–N (S1–S11) are samples which were S based-ELISA positive with OD ≥ 0.6 and O–R (S12–S15) with OD between 0.495 and <0.6.

Fig. 5

Detection of antibodies against native HCoV-HKU1 S protein expressed in BHK-21 cells by indirect immunofluorescent microscopy. Sera A and B (C1–2) are S-based ELISA negative samples. Serum C (S0) was taken from a patient who had recovered from HKU1 infection. Sera D–N are samples (S1–S11) which were S-based ELISA positive with OD ≥ 0.6 and O–R (S12–S15) with OD between 0.495 and <0.6.

Fig. 6

Seroprevalence of different age groups in HK-SAR were determined by presence of antibodies specific to recombinant HCoV-HKU1 S-based ELISA. Percentage positive above ELISA and neutralization antibodies cutoffs determined previously at OD450 >0.495 and 0.6 among various age groups were shown in the table.