key: cord-0992378-habpt92r
authors: Jagtap, Suraj; K, Ratnasri; Valloly, Priyanka; Sharma, Rakhi; Maurya, Satyaghosh; Gaigore, Anushree; Ardhya, Chitra; Biligi, Dayananda S.; Desiraju, Bapu Koundinya; Natchu, Uma Chandra Mouli; Saini, Deepak Kumar; Roy, Rahul
title: Evaluation of spike protein antigens for SARS-CoV-2 serology
date: 2021-06-29
journal: J Virol Methods
DOI: 10.1016/j.jviromet.2021.114222
sha: 4204ced3883e81c3d7ecbb3f92690293b644badd
doc_id: 992378
cord_uid: habpt92r

BACKGROUND: Spike protein domains are being used in various serology-based assays to detect prior exposure to SARS-CoV-2 virus. However, there has been limited comparison of antibody titers against various spike protein antigens among COVID-19 infected patients. METHODS: We compared four spike proteins (RBD, S1, S2 and a stabilized spike trimer (ST)) representing commonly used antigens for their reactivity to human IgG antibodies using indirect ELISA in serum from COVID-19 patients and pre-2020 samples. ST ELISA was also compared against the EUROIMMUN IgG ELISA test. Further, we estimated time appropriate IgG and IgA seropositivity rates in COVID-19 patients using a panel of sera samples collected longitudinally from the day of onset of symptoms (DOS). RESULTS: Among the four spike antigens tested, the ST demonstrated the highest sensitivity (86.2%; 95% CI: 77.8-91.7%), while all four antigens showed high specificity to COVID-19 sera (94.7-96.8%). 13.8% (13/94) of the samples did not show seroconversion in any of the four antigen-based assays. In a double-blinded head-to-head comparison, ST based IgG ELISA displayed a better sensitivity (87.5%, 95%CI: 76.4-93.8%) than the EUROIMMUN IgG ELISA (67.9%, 95% CI: 54.8-78.6%). Further, in ST-based assays, we found 48% and 50% seroconversion in the first six days (from DOS) for IgG and IgA antibodies, respectively, which increased to 84% (IgG) and 85% (IgA) for samples collected ≥22 days from DOS. CONCLUSIONS: Comparison of spike antigens demonstrates that spike trimer protein is a superior option as an ELISA antigen for COVID-19 serology.

COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has already crossed 178 million detected cases and over 3.8 million deaths worldwide till date (Dong et al., 2020) . As the pandemic continues, we need accurate and sensitive tests to assess the prevalence, disease burden and the level of population immunity against the virus.

With the introduction of multiple vaccines and several ongoing vaccine trials, identifying prior exposure or immunogenicity of the vaccine in individuals becomes critical to the development of vaccination and public health strategies.

Nucleic acid-based tests that detect viral RNA are widely used to diagnose active infection in SARS-CoV-2 infected individuals (Mathuria et al., 2020; Sethuraman et al., 2020) . In contrast, immunological tests like serological assays detect the level of antibody response in humans to the infection in symptomatic as well as the large fraction of asymptomatic infections (Galipeau et al., 2020; Long et al., 2020; Milani et al., 2020; Oved et al., 2020) . Immuno-assays detect antigenspecific IgA, IgM and IgG immunoglobulins (antibodies) from body fluids like serum or plasma. Viral antigen-specific antibodies can be detected in SARS-CoV-2 exposed individuals within 5-12 days post-onset of symptoms (POS) for IgM and IgA antibodies and 14 days for IgG antibodies (Guo et al., 2020; Zhao et al., 2020) . IgG antibodies are long-lived, detectable for up to 12 months, making them recent and long-term markers of exposure to SARS-CoV-2 compared to short-lived IgA and IgM (Dan et al., 2021; Laing et al., 2021) . Serological assays with nucleocapsid or spike (S) protein of SARS-CoV-2 as capturing antigen have been widely developed and reported, as these antigens are highly immunogenic Premkumar et al., 2020) . Nucleocapsid based ELISA assays have shown to be less specific, contributing to false positive results (Katz et al., 2020; Yamaoka et al., 2020) . The spike protein decorates the exterior of SARS-CoV-2 virus that helps the virus to bind to the ACE2 receptors on the host cell membrane, promotes fusion of the viral membrane with the host cell and thereby enables the entry of the viral genome into the host cell cytoplasm  J o u r n a l P r e -p r o o f efficacy (Suthar et al., 2020; Wajnberg et al., 2020) . The spike glycoprotein is a clove-shaped trimeric protein with each unit consisting of the S1 head and the S2 stalk. The Receptor Binding Domain (RBD) of the S1 head is responsible for binding to the ACE2 receptor on the cellular membrane, initiating cell entry Walls et al., 2020) . Due to the large size of the S protein and its highly hydrophobic S2 region, traditional mammalian expression systems produce low levels of recombinant protein. Additionally, absence of post-translational modifications in bacterial expression systems precludes the expression of this glycosylated protein in E. coli. Capture antigens used for serological assays should be easy to express and purify, with high yield and stability. Recently, the prefusion state of SARS-CoV-2 spike trimer (ST) protein was stabilized by the addition of 6 prolines that improved thermal stability and expression yield in mammalian cell suspension culture, making it a promising antigen for SARS-CoV-2 antibody assays (Hsieh et al., 2020) .

In this study, we evaluated the ST protein as a potential capture antigen for ELISA and compared it with different subunits of S protein, namely, S1, S2 and RBD to assess IgG antibody titers in SARS-CoV-2 positive and pre-pandemic sera. We also used ST protein to elucidate IgG and IgA antibody response dynamics with time-stratified samples (≤6, 7-14, 15-21 and ≥22 days POS).

Further, we benchmarked the ST protein ELISA against an FDA approved (EUROIMMUN) serology ELISA kit.

For COVID-19 samples, 1-2 ml of blood was drawn from patients who had tested positive for were collected, and assays were performed.

The plasmids for RBD (pCAGGS vector containing the human codon-optimized RBD (amino acids Head-to-head comparison of ST ELISA and EUROIMMUN Anti-SARS-CoV-2 (IgG) ELISA (S1 protein-based serology kit approved by FDA and ICMR, EI 2606-9601 G (EUROIMMUN, 2020)) was performed in a double-blind format where the experimenters were blind to the RT-PCR and seropositivity results. EUROIMMUN ELISA was performed as per the manufacturer's instructions.

All statistical analyses and visualization were done using custom-written python codes and GraphPad Prism software (v8.4.3). Unpaired two-tailed Student's t-test was used to compare the COVID-19 positive and negative groups. Confidence intervals were calculated using Wilson/Brown's method (Brown et al., 2001) .

We compared four different spike protein antigens (S1, S2, RBD and ST) that represent different protein segments commonly being used to evaluate serum reactivity among SARS-CoV-2 patients (Figure 1a) . 94 COVID-19 samples collected ≥15 days from DOS/RT, and 94 control samples were tested for the presence of spike-specific IgG antibodies (Figure 1b) . We noted that J o u r n a l P r e -p r o o f in-house purified RBD and ST performed better than commercially procured S1 and S2 in terms of sensitivity and intensity of the positive sample signal. ST showed the highest sensitivity (86.2%) followed by RBD (69.9%), while S1 and S2 domains showed very low sensitivity (51.5% and 50.0%, respectively) ( 

We performed ELISA assays for spike specific IgG and IgA antibodies in sera collected at different shown median time for seroconversion based on spike specific IgG antibodies to be 14 days POS Wölfel et al., 2020; Zhao et al., 2020) . A high correlation (PCC = 0.76, Figure 3d Some individuals did not show detectable spike-specific antibodies (16% for IgG, 15% for IgA) even after 21 days from DOS. This could be due to asymptomatic/mild infection which has been reported to display low antibody titers (Long et al., 2020) . We cannot rule-out false-positive PCR results contributing to some of these cases.

The trimeric spike protein from SARS-CoV-2 is critical for cellular entry and is prominently displayed on the virus. Our results revealed that ST protein displayed better reactivity to COVID-J o u r n a l P r e -p r o o f larger number of accessible antibody epitopes on ST. While we did not perform neutralization assays, previous studies have shown that the antibodies against RBD are strongly correlated with neutralization of the virus Suthar et al., 2020; Wajnberg et al., 2020) . Several other regions of the Spike protein, including a region away from receptor binding site , S1 domain (Hoffmann et al., 2020) and S2 domain (Duan et al., 2005; Elshabrawy et al., 2012; Xia et al., 2020) are targets of neutralizing antibodies. Therefore, a high ELISA signal against the ST protein is suggestive of higher levels of a broader spectrum of virus-neutralizing antibodies.

Purification of the trimeric spike protein (ST) from mammalian cell culture has generally been challenging compared to the smaller RBD segment Klumpp-Thomas et al., 2021) . Recent development of pre-fusion stabilized spike variant (HexaPro) seems to resolve this issue (Hsieh et al., 2020) . We indeed recovered > 10 mg/L ST protein consistently in our preparations. However, it was not clear if this stabilized ST protein with six proline mutations is sufficiently antigenic against COVID-19 infection induced antibodies. Our results suggests that HexaPro variant of the spike protein indeed folds and maintains the original epitopes comparable to the spike on the SARS-CoV-2 virus.

IgG and IgA antibody dynamics show seroconversion in about half of the patients within 6 days POS (also reported earlier, Wölfel et al., 2020; Zhao et al., 2020) ). Considering the low sensitivity of many existing rapid antigen tests (Gremmels et al., 2020; ICMR, 2020; Scohy et al., 2020) , a combination of the rapid antigen detection with antibody tests can be employed to increase the detection efficiency of COVID-19 cases during early infection.

Some COVID-19 RT-PCR positive samples did not show reactivity against any of the four antigens. Several other studies have reported limited seroconversion at the time of sera collection (Brochot et al., 2020; Long et al., 2020; Staines et al., 2020) . Nevertheless, highly sensitive serology assays can be critical in determining the sero-prevalence in a community as well as help J o u r n a l P r e -p r o o f

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This research has been conducted with the contribution of NCR Biotech Science Cluster