Hybrid and herd immunity 6 months after SARS-CoV-2 publicity in people from a neighborhood remedy program


This observational study involved 79 participants from 15 families who were randomly invited from different metropolitan areas of Bangkok. At least one SARS-CoV-2 infected patient in each family had to be registered in a Bangkok Home Health Care Service database between August 1 and August 31, 2021. In addition, there had to be at least 1 asymptomatic close contact with negative antigen living in the same accommodation in accordance with the national health policy regulation. 34 cases were people who had recovered from SARS-CoV-2 at least 4 weeks prior to enrollment, while 45 cases were close contacts. The calculated sample size was 90 cases based on the prevalence of asymptomatic SARS-CoV-2 among close contacts reported in the Chinese population4 with an 80 percent probability of detecting asymptomatic infections. Peripheral blood (15 mL) was collected after obtaining consent from the participants. This study was approved by the Ethics Committee of Ramathibodi Hospital, Mahidol University (MURA2021/923) and Bangkok Hospital (BHQ-IRB 2021-11-34) in accordance with the Declaration of Helsinki, the Belmont Report, the CIOM Guidelines and the International Conference on Approves Harmonization in Good Clinical Practice (ICH-GCP).

Materials and Reagents

A FACSLyric™ (Becton Dickinson, USA) flow cytometer was used for cytokine detection and Beckman Coulter’s FC 500 series (Beckman Coulter, USA) was used for CBC determination. The Alinity and ARCHITECT systems were used for the IgG values ​​against SARS-CoV-2 and the EUROIMMUN Analyzer I for the ELISA reader. The incubator, centrifuge, vortex mixer and automatic cell counter were from Thermo Fisher Scientific, Inc. (USA). Phosphate buffered saline (PBS) pH 7.4 was purchased from Sigma-Aldrich (USA), heparin and EDTA tubes were purchased from Becton Dickinson (USA), and RPMI 1640 medium was purchased from Life Technologies (USA ) based.

The Enzyme-Linked Immunosorbent Spot (ELISpot) Assay and T-SPOT

Fresh heparinized whole blood samples (10 ml) from volunteers were isolated onto peripheral blood mononuclear cells (PBMCs) using SepMate PBMC isolation tubes (STEMCELL Technologies Inc., Canada). The heparinized blood was diluted with RPMI medium (1:1) in SepMate PBMC isolation tubes and centrifuged at 1700 g at 20°C for 20 min. The separated PBMCs were washed twice with PBS and recentrifuged at 500 xg for 5 min at 4°C. PBMCs (2.5 x 10 5 cells) were added to 96-well plates pre-coated with an anti-IFN-g antibody of the T-SPOT® COVID test (Oxford Immunotec, Ltd., UK). The plate stimulated each four-well sample with two antigens against the spike protein (S) and nucleocapsid protein (N), the membrane glycoprotein (M), and the ORF1ab region of the RNA-dependent RNA (O) of SARS- CoV-2 alpha variant; Phytohemagglutinin (PHA) and medium alone were used as positive and negative controls, respectively. Plates were maintained overnight at 37°C in a humidified 5% CO2 atmosphere, washed with phosphate-buffered saline, and developed using an anti-IFN-g antibody conjugate and substrate to detect the presence of secreted IFN-g . Spotting cells (SFCs) were counted with an automated ELISpot reader (CTL Analyzers, Cleveland, OH, USA). Less than 10 SFCs per 250,000 cells represented normal background according to the manufacturer’s recommendations, which was comparable to the bottom quartile of SFCs in SARS-CoV-2 patients in this study.

SARS-CoV-2 IgG levels in the receptor-binding domain (RBD)

Human EDTA plasma samples were measured to quantify IgG antibodies against the spike receptor binding domain (RBD) of SARS-CoV-2 using the SARS-CoV-2 IgG II Quant Assay based on the Alinity and ARCHITECT I systems was used. The chemiluminescent response was calculated as relative light units (RLU) and expressed as calculated index (S/C). The SARS-CoV-2 IgG-II Quant Assay cutoff was 50 AU/mL and values ​​above 50 AU/mL were interpreted as positive.

ELISA for the detection of SARS-CoV-2 specific neutralizing antibodies

Human EDTA plasma samples were diluted 1:5 in sample buffer following the manufacturer’s instructions for the Euroimmun SARS-CoV-2 NeutraLISA (Euroimmun AG, Lübeck, Germany). Briefly, 100 μL of the diluted sample, control or blank was added per well and incubated at 37 °C for 1 h. The automatic machine washed the plate three times with washing buffer; then 100 μl of enzyme conjugate were added and incubated at RT for 30 min. After washing, 100 µL of substrate solution were added and the plate was incubated at RT for 15 min. Finally, 100 μL of stop solution were added per well and the absorbance at 450 nm was measured with a EUROIMMUN Analyzer I. The samples were analyzed in one replicate. Percent inhibition (% IH) was calculated as follows: 100% – [(extinction of sample × 100%)/extinction of blank]. Euroimmun recommends interpreting the results as follows: %IH< 20: negative; %IH >20 to< 35: borderline; and %IH >35: positive.

Statistical analysis

Descriptive results are presented as medians (interquartile ranges) and percentages ± standard deviations. Corresponding inferential comparisons were performed using the t-test or Mann-Whitney U-test and the correlation analysis was calculated using GraphPad Prism Version 9.4.0 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com.

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