Background Understanding the longitudinal trajectory of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies is crucial for diagnosis of prior infection and predicting future immunity. Methods We conducted a longitudinal analysis of coronavirus disease 2019 convalescent patients, with neutralizing antibody assays and SARS-CoV-2 serological assay platforms using SARS-CoV-2 spike (S) or nucleocapsid (N) antigens. Results Sensitivities of serological assays in diagnosing prior SARS-CoV-2 infection changed with time. One widely used commercial platform that had an initial sensitivity of >95% declined to 71% at 81-100 days after diagnosis. The trajectories of median binding antibody titers measured over approximately 3-4 months were not dependent on the use of SARS-CoV-2 N or S proteins as antigen. The median neutralization titer decreased by approximately 45% per month. Each serological assay gave quantitative antibody titers that were correlated with SARS-CoV-2 neutralization titers, but S-based serological assay measurements better predicted neutralization potency. Correlation between S-binding and neutralization titers deteriorated with time, and decreases in neutralization titers were not predicted by changes in S-binding antibody titers. Conclusions Different SARS-CoV-2 serological assays are more or less well suited for surveillance versus prediction of serum neutralization potency. Extended follow-up should facilitate the establishment of appropriate serological correlates of protection against SARS-CoV-2 reinfection.

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Longitudinal Serological Analysis and Neutralizing Antibody Levels in Coronavirus Disease 2019 Convalescent Patients

Quantification of SARS-CoV-2 antibodies with eight commercially available immunoassays.

SARS-CoV-2 seroprevalence among the general population and healthcare workers in India, December 2020-January 2021.

Pseudoviruses are useful surrogates for highly pathogenic viruses because of their safety, genetic stability, and scalability for screening assays. Many different pseudovirus platforms exist, each with different advantages and limitations. Here we report our efforts to optimize and characterize an HIV-based lentiviral pseudovirus assay for screening neutralizing antibodies for SARS-CoV-2 using a stable 293T cell line expressing human angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). We assessed different target cells, established conditions that generate readouts over at least a two-log range, and confirmed consistent neutralization titers over a range of pseudovirus input. Using reference sera and plasma panels, we evaluated assay precision and showed that our neutralization titers correlate well with results reported in other assays. Overall, our lentiviral assay is relatively simple, scalable, and suitable for a variety of SARS-CoV-2 entry and neutralization screening assays.

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Establishment of a well-characterized SARS-CoV-2 lentiviral pseudovirus neutralization assay using 293T cells with stable expression of ACE2 and TMPRSS2.

Neutralizing antibodies targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) block severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry into cells via surface-expressed angiotensin-converting enzyme 2 (ACE2). We used a surrogate virus neutralization test (sVNT) and SARS-CoV-2 S protein-pseudotyped vesicular stomatitis virus (VSV) vector-based neutralization assay (pVNT) to assess the degree to which serum antibodies from coronavirus disease 2019 (COVID-19) convalescent patients interfere with the binding of SARS-CoV-2 S to ACE2. Both tests revealed neutralizing anti-SARS-CoV-2 S antibodies in the sera of ~90% of mildly and 100% of severely affected COVID-19 convalescent patients. Importantly, sVNT and pVNT results correlated strongly with each other and to the levels of anti-SARS-CoV-2 S1 IgG and IgA antibodies. Moreover, levels of neutralizing antibodies correlated with the duration and severity of clinical symptoms but not with patient age. Compared to pVNT, sVNT is less sophisticated and does not require any biosafety labs. Since this assay is also much faster and cheaper, sVNT will not only be important for evaluating the prevalence of neutralizing antibodies in a population but also for identifying promising plasma donors for successful passive antibody therapy.

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Low serum neutralizing anti-SARS-CoV-2 S antibody levels in mildly affected COVID-19 convalescent patients revealed by two different detection methods.

Severe acute respiratory syndrome coronavirus 2 infection and development of coronavirus disease 2019 presents a major health care challenge of global dimensions. Laboratory diagnostics of infected patients, and the assessment of immunity against severe acute respiratory syndrome coronavirus 2, presents a major cornerstone in handling the pandemic. Currently, there is an increase in demand for antibody testing and a large number of tests are already marketed or are in the late stage of development. However, the interpretation of test results depends on many variables and factors, including sensitivity, specificity, potential cross-reactivity and cross-protectivity, the diagnostic value of antibodies of different isotypes, and the use of antibody testing in identification of acutely ill patients or in epidemiological settings. In this article, the recently established COVID-19 Task Force of the German Society for Clinical Chemistry and Laboratory Medicine (DGKL) addresses these issues on the basis of currently available data sets in this rapidly moving field.

Oliver Frey

Papers

SARS-CoV-2 antibody testing-questions to be asked.