Serological assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), March 2020.

BackgroundThe ongoing coronavirus disease (COVID-19) pandemic has major impacts on health systems, the economy and society. Assessing infection attack rates in the population is critical for estimating disease severity and herd immunity which is needed to calibrate public health interventions. We have previously shown that it is possible to achieve this in real time to impact public health decision making.AimOur objective was to develop and evaluate serological assays applicable in large-scale sero-epidemiological studies.MethodsWe developed an ELISA to detect IgG and IgM antibodies to the receptor-binding domain (RBD) of the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluated its sensitivity and specificity in combination with confirmatory microneutralisation (MN) and 90% plaque reduction neutralisation tests (PRNT90) in 51 sera from 24 patients with virologically confirmed COVID-19 and in age-stratified sera from 200 healthy controls.ResultsIgG and IgM RBD ELISA, MN and PRNT90 were reliably positive after 29 days from illness onset with no detectable cross-reactivity in age-stratified controls. We found that PRNT90 tests were more sensitive in detecting antibody than MN tests carried out with the conventional 100 tissue culture infectious dose challenge. Heparinised plasma appeared to reduce the infectivity of the virus challenge dose and may confound interpretation of neutralisation test.ConclusionUsing IgG ELISA based on the RBD of the spike protein to screen sera for SARS-CoV-2 antibody, followed by confirmation using PRNT90, is a valid approach for large-scale sero-epidemiology studies.

Long-term persistence of SARS-CoV-2 neutralizing antibody responses after infection and estimates of the duration of protection.

BACKGROUND: The duration of immunity in SARS-CoV-2 infected people remains unclear. Neutralizing antibody responses are the best available correlate of protection against re-infection. Recent studies estimated that the correlate of 50% protection from re-infection was 20% of the mean convalescent neutralizing antibody titre. METHODS: We collected sera from a cohort of 124 individuals with RT-PCR confirmed SARS-CoV-2 infections from Prince of Wales Hospital, Princess Margaret Hospital, Queen Elizabeth Hospital and Queen Mary Hospitals of the Hospital Authority of Hong Kong, for periods up to 386 days after symptom onset and tested these for antibody to SARS-CoV-2 using 50% virus plaque reduction neutralization tests (PRNT50), surrogate neutralization tests and spike receptor binding domain (RBD) binding antibody. Patients were recruited from 21 January 2020 to 16 February 2021 and follow-up samples were collected until 9th March 2021. FINDINGS: Because the rate of antibody waning slows with time, we fitted lines of decay to 115 sera from 62 patients collected beyond 90 days after symptom onset and estimate that PRNT50 antibody will remain detectable for around 1,717 days after symptom onset and that levels conferring 50% protection will be maintained for around 990 days post-symptom onset, in symptomatic patients. This would potentially be affected by emerging virus variants. PRNT titres wane faster in children. There was a high level of correlation between PRNT50 antibody titers and the % of inhibition in surrogate virus neutralization tests. INTERPRETATION: The data suggest that symptomatic COVID-19 disease is followed by relatively long-lived protection from re-infection by antigenically similar viruses. FUNDING: Health and Medical Research Fund, Commissioned research on Novel Coronavirus Disease (COVID-19) (Reference Nos. COVID190126 and COVID1903003) from the Food and Health Bureau and the Theme-based Research Scheme project no. T11-712/19-N, the University Grants Committee of the Hong Kong SAR Government.

The SARS-CoV-2 antibody landscape is lower in magnitude for structural proteins, diversified for accessory proteins and stable long-term in children.

Background: Children are less clinically affected by SARS-CoV-2 infection than adults with the majority of cases being mild or asymptomatic and the differences in infection outcomes are poorly understood. The kinetics, magnitude and landscape of the antibody response may impact the clinical severity and serological diagnosis of COVID-19. Thus, a comprehensive investigation of the antibody landscape in children and adults is needed. Methods: We tested 254 plasma from 122 children with symptomatic and asymptomatic SARS-CoV-2 infections in Hong Kong up to 206 days post symptom onset, including 146 longitudinal samples from 58 children. Adult COVID-19 patients and pre-pandemic controls were included for comparison. We assessed antibodies to a 14-wide panel of SARS-CoV-2 structural and accessory proteins by Luciferase Immunoprecipitation System (LIPS). Findings: Children have lower levels of Spike and Nucleocapsid antibodies than adults, and their cumulative humoral response is more expanded to accessory proteins (NSP1 and Open Reading Frames (ORFs)). Sensitive serology using the three N, ORF3b, ORF8 antibodies can discriminate COVID-19 in children. Principal component analysis revealed distinct serological signatures in children and the highest contribution to variance were responses to non-structural proteins ORF3b, NSP1, ORF7a and ORF8. Longitudinal sampling revealed maintenance or increase of antibodies for at least 6 months, except for ORF7b antibodies which showed decline. It was interesting to note that children have higher antibody responses towards known IFN antagonists: ORF3b, ORF6 and ORF7a. The diversified SARS-CoV-2 antibody response in children may be an important factor in driving control of SARS-CoV-2 infection.

SARS-CoV-2 specific T cell responses are lower in children and increase with age and time after infection.

SARS-CoV-2 infection of children leads to a mild illness and the immunological differences with adults remains unclear. We quantified the SARS-CoV-2 specific T cell responses in adults and children (<13 years of age) with RT-PCR confirmed asymptomatic and symptomatic infection for long-term memory, phenotype and polyfunctional cytokines. Acute and memory CD4+ T cell responses to structural SARS-CoV-2 proteins significantly increased with age, whilst CD8+ T cell responses increased with time post infection. Infected children had significantly lower CD4+ and CD8+ T cell responses to SARS-CoV-2 structural and ORF1ab proteins compared to infected adults. SARS-CoV-2-specific CD8+ T cell responses were comparable in magnitude to uninfected negative adult controls. In infected adults CD4+ T cell specificity was skewed towards structural peptides, whilst children had increased contribution of ORF1ab responses. This may reflect differing T cell compartmentalisation for antigen processing during antigen exposure or lower recruitment of memory populations. T cell polyfunctional cytokine production was comparable between children and adults, but children had a lower proportion of SARS-CoV-2 CD4+ T cell effector memory. Compared to adults, children had significantly lower levels of antibodies to ß-coronaviruses, indicating differing baseline immunity. Total T follicular helper responses was increased in children during acute infection indicating rapid co-ordination of the T and B cell responses. However total monocyte responses were reduced in children which may be reflective of differing levels of inflammation between children and adults. Therefore, reduced prior ß-coronavirus immunity and reduced activation and recruitment of de novo responses in children may drive milder COVID-19 pathogenesis.

Chronic hepatitis C genotype 6 responds better to pegylated interferon and ribavirin combination therapy than genotype 1.

BACKGROUND AND AIMS: Chronic hepatitis C genotype 6 is common in Hong Kong, especially among i.v. drug abusers. Responses of these patients to combination of pegylated interferon and ribavirin treatment were inconsistent and the numbers of patients involved in previous studies were small. We performed a retrospective study to compare the therapeutic responses of this regimen in patients infected with genotype 6 and genotype 1. METHODS: Seventy patients with either genotype 6 or genotype 1 were recruited. Both groups received 800-1200 mg of ribavirin daily plus either 180 mg of pegylated alpha-interferon-2a or 1.5 mg/kg pegylated alpha-interferon-2b weekly for 48 weeks. Their responses to treatments were compared. RESULTS: The early virological response to combination therapy of patients with genotype 6 was significantly better than that of genotype 1 (88.6% vs 74.3%, P = 0.03). Significant difference was also identified in the end of treatment response of the two genotypes (60% vs 81.4% for genotype 1 and 6, respectively; P = 0.005). The sustained virological response (SVR) to treatment in patients with genotype 6 was also significantly superior to that of patients with genotype 1 (75.7% vs 57.1%, P = 0.02). Multiple logistic regression analysis demonstrated that age of 55 years or less, genotypes of hepatitis C virus, liver biopsy staging and baseline hepatitis C virus RNA of 200,000 IU/mL or less were independent predictors for better SVR in this cohort. CONCLUSION: Patients with chronic hepatitis C genotype 6 respond better to pegylated interferon and ribavirin combination treatment than patients with genotype 1.