Serologic Responses in Healthy Adult with SARS-CoV-2 Reinfection, Hong Kong, August 2020

In March 2020, mild signs and symptoms of coronavirus disease developed in a healthy 33-year-old man in Hong Kong. His first infection did not produce virus neutralizing antibodies. In August, he had asymptomatic reinfection, suggesting that persons without a robust neutralizing antibody response might be at risk for reinfection.

PRNT antibodies were detected as previously described (1). Briefly, serial dilutions of each serum sample were incubated with 30-40 plaque-forming units of viruses for 1 h at 37°C.
The virus-serum mixtures were added onto pre-formed Vero E6 cell monolayers and incubated for 1 h at 37°C in 5% CO2 incubator. The cell monolayer was then overlaid with 1% agarose in cell culture medium. After 3 d of incubation, the plates were fixed and stained. Antibody titers were defined as the highest serum dilution that resulted in >50% (PRNT50) reduction in the number of virus plaques.

Surrogate Virus Neutralization Assay
Neutralizing antibodies were also tested by the SARS-CoV-2 surrogate virus neutralization test kits (GeneScript USA, Inc, New Jersey) according to the manufacturer's instructions (2). Briefly, the test sera (60 µL), the positive and negative controls were diluted at 1:10 and mixed with an equal volume of horseradish peroxidase (HRP) conjugated SARS-CoV-2 spike receptor binding domain (RBD) protein and incubated for 30 min at 37°C. Then, 100 µL of each mix was added to the wells on the microtiter plate coated with ACE-2 receptor, the plate was sealed and incubated at 37°C for 15 min. The plates were then washed with wash-solution, Page 2 of 7 tapped dry and 100 µL of 3,3′,5,5′-Tetramethylbenzidine (TMB) solution was added to each well and incubated in the dark at room temperature for 15 min. The reaction was stopped by addition of 50 µL of Stop Solution to each well and the absorbance read at 450 nm in an ELISA reader.
Assuming the positive and negative controls gave the recommended OD450 values, the % inhibition of each serum was calculated as (1 -OD value of the sample/OD value of the negative control) × 100. An inhibition of >20% is regarded as a positive result while that <20% is negative (2).

Luciferase Immunoprecipitation (LIPS) Assay
The LIPS assay was initially described by Burbelo et al. (3) adapted to SARS-CoV-2 by us as previously described (4). Briefly, Renilla luciferase tagged SARS-CoV-2 N antigens were produced from COS1 cells, and antigen equalized to 10 7 luciferase units for each serological test.
Serum (heat inactivated and diluted 1:100 in Buffer A (50 mM Tris, pH 7.5, 100 mM NaCl, 5 mM MgCl2, 1% Triton X-100)) was incubated with 10 7 LU of (Ruc)-N antigen for 1 h with shaking at 800 rpm. Ultralink protein A/G beads were added to the (Ruc)-antigen and serum mixture in a 96-deep-well polypropylene microtiter plate and incubated for 1 h with shaking at 800 rpm. The entire volume was then transferred into HTS plates and washed 10 times with Buffer A, and then twice with PBS. The plate was read using QUANTI-Luc Gold substrate Experimental controls include no-serum blank wells with (Ruc)-antigens and negative control serum from age-matched noninfected patient plasma collected prior to the COVID-19 pandemic (n = 20). The background corresponds to the LU signal from each Ruc-fusion antigen with protein A/G and substrate with no serum. The cutoff limits were derived from the mean value plus 3 × SD of the negative controls.

Virus Full-Genome Sequencing
We used the methods previously described by us (5). Briefly, virus genome was reverse transcribed with multiple gene-specific primers targeting different regions of the viral genome.
The synthesized cDNA was then subjected to multiple overlapping 2-kb PCRs for full-genome amplification. PCR amplicons obtained from the same specimen were pooled and sequenced using MiSeq sequencing platform (Illumina). Sequencing library was prepared by Nextera XT Page 3 of 7 DNA library prep Kit (Illumina) following standard protocol. Generated sequencing reads were mapped to a reference virus genome by BWA (6), and genome consensus was generated by Geneious version 11.1.4 (https://www.geneious.com).