Assessment of antibody dynamics and neutralizing activity using serological assay after SARS-CoV-2 infection and vaccination

The COVID-19 antibody test was developed to investigate the humoral immune response to SARS-CoV-2 infection. In this study, we examined whether S antibody titers measured using the anti-SARS-CoV-2 IgG II Quant assay (S-IgG), a high-throughput test method, reflects the neutralizing capacity acquired after SARS-CoV-2 infection or vaccination. To assess the antibody dynamics and neutralizing potency, we utilized a total of 457 serum samples from 253 individuals: 325 samples from 128 COVID-19 patients including 136 samples from 29 severe/critical cases (Group S), 155 samples from 71 mild/moderate cases (Group M), and 132 samples from 132 health care workers (HCWs) who have received 2 doses of the BNT162b2 vaccinations. The authentic virus neutralization assay, the surrogate virus neutralizing antibody test (sVNT), and the Anti-N SARS-CoV-2 IgG assay (N-IgG) have been performed along with the S-IgG. The S-IgG correlated well with the neutralizing activity detected by the authentic virus neutralization assay (0.8904. of Spearman’s rho value, p < 0.0001) and sVNT (0.9206. of Spearman’s rho value, p < 0.0001). However, 4 samples (2.3%) of S-IgG and 8 samples (4.5%) of sVNT were inconsistent with negative results for neutralizing activity of the authentic virus neutralization assay. The kinetics of the SARS-CoV-2 neutralizing antibodies and anti-S IgG in severe cases were faster than the mild cases. All the HCWs elicited anti-S IgG titer after the second vaccination. However, the HCWs with history of COVID-19 or positive N-IgG elicited higher anti-S IgG titers than those who did not have it previously. Furthermore, it is difficult to predict the risk of breakthrough infection from anti-S IgG or sVNT antibody titers in HCWs after the second vaccination. Our data shows that the use of anti-S IgG titers as direct quantitative markers of neutralizing capacity is limited. Thus, antibody tests should be carefully interpreted when used as serological markers for diagnosis, treatment, and prophylaxis of COVID-19.


Introduction
Coronavirus disease 2019 (COVID- 19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is currently an endemic worldwide [1].Reverse transcription polymerase chain reaction (RT-PCR) is the gold standard test for the diagnosis of SARS-CoV-2 infection [2,3].However, false negative results of RT-PCR can be caused by suboptimal primer design, imperfect RNA extraction techniques, or lower volumes of applied virus [4].Furthermore, because the RNA concentration of SARS-CoV-2 declines from 1-2 weeks after symptom onset, the detection ratio of RT-PCR is also decreased [5].On the other hand, because seroconversion of SARS-CoV-2 occurs between one to two weeks after symptom onset, the serological antibody tests' detection rate of specific antibody gradually increases [6].
Many of the commercial antibody tests can specifically detect immunoglobulins, such as IgG and IgM, binding against the nucleocapsid (N) protein and the receptor-binding domain (RBD) in the spike (S) protein of SARS-CoV-2 [7].They are used as an adjunct to RT-PCR for COVID-19 diagnosis [8].The RBD directly binds to angiotensin-converting enzyme 2 (ACE2), a host cell receptor that mediates attachment of SARS-CoV-2 [9].Since 90% of the neutralizing activity against SARS-CoV-2 targets RBD [10,11], anti-RBD antibodies have the potential to neutralize viral entry into cells and could be a marker of protective immune response against SARS-CoV-2 infection [12,13].Assays that detect neutralizing activity are recognized as reliable, but authentic virus neutralization assays are restricted to Biosafety Level 3 (BSL3) facilities, and a few are available.Even the surrogate virus neutralization test (sVNT), based on enzyme-linked immunoassay (ELISA), is time-consuming and has low-throughput.Therefore, high-throughput, widely available quantitative S-IgG has been developed.
In this study, we evaluated whether the antibody titers using the Abbott SARS-CoV-2 IgG II Quant assay, an automated chemiluminescent immunoassay for detecting SARS-CoV-2 S specific antibodies, accurately reflects the antibody dynamics and neutralizing activity following SARS-CoV-2 infection and vaccination using sVNT and full-scale virus neutralization assays.

Ethics statement
This study complied with all relevant national regulations and institutional policies.It was conducted in accordance with the tenets of the Declaration of Helsinki and was approved by the Institutional Review Board (IRB) at Juntendo University Hospital (IRB # 20-036).The need for informed consent from individual patients was waived because all samples were deidentified in line with the Declaration of Helsinki.Informed consent was obtained from HCWs (IRB # M20-0089-M01).

Clinical backgrounds
This study was conducted at Juntendo University Hospital in Japan and included a total of 457 blood serum samples from 253 individuals.Three hundred and twenty-five samples were collected from 128 COVID-19 patients confirmed by RT-PCR between March and September

Serologic testing for SARS-CoV-2
Anti-S SARS-CoV-2 IgG II Quant assay (S-IgG) was performed on the Abbott Alinity i platform (Abbott Laboratories, Chicago, IL, USA) according to the manufacturer's instructions.The assay is based on the chemiluminescent microparticle immunoassay (CMIA) for qualitative detection of anti-S IgG in human serum/plasma against the S glycoprotein on the surface of SARS-CoV-2 [17].
Anti-N SARS-CoV-2 IgG assay (N-IgG) targets N protein and was performed on the Abbott Alinity i platform according to the manufacturer's instructions.The assay is based on the CMIA for semi-quantitative assessment of anti-N IgG.The resulting chemiluminescence in relative light units indicates the strength of the response, which reflects each specific antibody present.Results from the quantitative S-IgG are reported as arbitrary units (AU) per milliliter, and values equal to the cutoff of 50 AU/mL or greater were classified as positive [18].Results from the semi-quantitative N-IgG are reported as index values, and the manufacturer's suggested positive cutoff point of 1.40 was used [19,20].

Virus neutralization assay
The authentic virus neutralization assay has been performed as described previously [21].The SARS-CoV-2 ancestral strain WK-521 (lineage A, GISAID ID: EPI_ISL_408667) was used for the authentic virus neutralization assay which has been performed at the National Institute of Infectious Diseases (NIID) with ethics approval by the medical research ethics committee of NIID for the use of human subjects (#1178).Briefly, serially diluted serum samples (2-fold serial dilutions starting at 1:5 dilution, diluted with high glucose Dulbecco's Modified Eagle Medium supplemented with 2% Fetal Bovine Serum and 100 U/mL penicillin/streptomycin, from Fujifilm Wako Pure Chemicals, Japan) were mixed with the virus from 100 Median Tissue Culture Infectious Dose (TCID50) and incubated at 37˚C for 1 hour.The mixture was subsequently incubated with VeroE6/TMPRSS2 cells (JCRB1819, JCRB Cell Bank, Japan) and seeded in 96-well flat-bottom plates for 4-6 days at 37˚C in a chamber supplied with 5% CO 2 .The cells were then fixed with 20% formalin (Fujifilm Wako Pure Chemicals) and stained with crystal violet solution (Sigma-Aldrich, St Louis, MO).Each sample was assayed in 2-4 wells and the average cut-off dilution index of > 50% cytopathic effect was presented as a neutralizing titer.Neutralizing titer of the sample below the detection limit (1:5 dilution) was set as 2.5.Neutralizing antibody titer of < 5 is considered negative and � 5 was considered positive.
The surrogate virus neutralizing antibody test (sVNT) has been performed using the Gen-Script cPass 1 SARS-CoV-2 Antibody Detection Kit, a blocking enzyme-linked immunosorbent assay (GenScript, Piscataway, New Jersey, USA), following the company's instructions.Briefly, the samples and controls were pre-incubated with the horseradish peroxidase (HRP)labeled recombinant RBD proteins and the mixture was added to a capture plate pre-coated with the hACE2 proteins.After the complex of neutralizing antibody binding RBD-HRP was removed by washing, the wells were read at 450 nm in a microtiter plate reader.The percent signal inhibition for the detection of neutralizing antibodies were calculated as follows:

Statistical analysis
Data analysis was carried out using GraphPad Prism software (version 9.0.1;San Diego, CA, USA) and R software (version 4.1.0).Titers of antibodies were log-transformed before statistical analyses.Analysis between antibody titer and neutralization test was performed using the Spearman's rank correlations coefficient.When analyzing statistical differences between two or more experimental groups, one-way analysis of variance (ANOVA) and Tukey multiple comparison post hoc analysis were used.The Wilcoxon signed rank test was used in the test analysis for the two experimental groups.
The following notation was used to show statistical significance: * p value < 0.01, and ** p value < 0.001.
For longitudinal assessment, the kinetics in the emergence of anti-S IgG and sVNT titers were determined for the S (severe and critical) and M (mild and moderate) groups using a nonlinear mixed effects model.The models were fitted to a four-parameter logistic function, with a constrained lower asymptote set to the limit of detection, the inflection point, a scale parameter, and the upper asymptote for Group S and Group M. A comparison between Group S and Group M was conducted in a Z test using the estimations.

Correlations between anti-S IgG titer and neutralizing activities
We first compared the results of S-IgG to sVNT and then S-IgG to the neutralizing activity.As shown in Fig 1, the results of 176 samples demonstrated a strong linear correlation between S-IgG and sVNT.The relationship between S-IgG and the authentic virus neutralizing assay also showed a liner correlation.We further confirmed a linear correlation between sVNT and the authentic virus neutralizing assay.Comparison of S-IgG and the authentic virus neutralizing assay revealed a positive percent agreement (PPA) of 96.8% and a negative percent agreement (NPA) of 92.3%.Similarly, a comparison of sVNT and the authentic virus neutralizing assay revealed a PPA of 97.6% and a NPA of 84.6%.However, when compared to the authentic virus neutralization assay, 4 samples (2.3%) tested for S-IgG and 8 samples (4.5%) tested for sVNT were inconsistent with the negative results for the neutralizing activity of the comparative method.On the other hand, 4 samples (2.3%) tested for S-IgG and 3 samples (1.7%) tested for sVNT were inconsistent with the positive results for the neutralizing activity of the comparative method.Samples with a titer � 20 in the authentic virus neutralizing assay were all positive in S-IgG and sVNT.

Longitudinal assessment of antibody titers in COVID-19 patients
To examine chronological changes in anti-S IgG and sVNT titers, we plotted these titers of inpatients whose antibodies were measured three or more times.Samples were collected up to 60 days after symptom onset to determine the antibodies' rate of change.The age distribution of each group was 50-90 years for Group S and 20-80 years for the Group M. Anti-S IgG and sVNT titers from patients of Group S and Group M were plotted against time from symptom onset and fitted (Fig 2).
Group S showed earlier increases in both anti-S IgG and sVNT titers than Group M. The 80% maximal response of anti-S IgG was achieved on day 17 for Group S and on day 23 for Group M. Similar kinetics were observed with sVNT, which achieved the 80% maximal response in Group S on day 15 and in Group M on day 23.No significant difference of maximal plateau value between Group S and Group M was observed for both anti-S IgG and sVNT (S-IgG: Group S, 7617.0,Group M, 4131.9;sVNT: Group S, 91.9, Group M, 91.0).

Distribution of anti-S IgG and sVNT titers after second vaccination
We then investigated S-IgG, sVNT, and N-IgG in 132 HCWs who received two doses of the vaccine.Because seropositive individuals with N-IgG are considered as previously infected with SARS-CoV-2 regardless of symptoms, the tested individuals were divided into four groups based on N-IgG results (positive/negative) and COVID-19 medical history.We observed that all tested individuals were seropositive with both S-IgG and sVNT.As shown in Fig 3, anti-S IgG and sVNT titers in N-IgG negative individuals with no medical history of COVID-19 were significantly lower compared to those with COVID-19 medical history and/ or N-IgG positive.The N-IgG positive individuals showed comparable anti-S IgG and sVNT titers to those with COVID-19 medical history.
Among these HCWs, we assessed the anti-S IgG and sVNT titers in breakthrough infection cases who were diagnosed with SARS-CoV-2 infection by RT-PCR after the second

Discussion
In this study, we investigated the serological of antibody dynamics and neutralizing potency following SARS-CoV-2 infection and post vaccination by comparing three quantitative assays with different principles for detection of antibodies to SARS-CoV-2.S-IgG is correlated well with both sVNT and the authentic virus neutralization assay with high PPA and NPA.However, when the authentic virus neutralization assay was used as the comparative method, the results of 2.3% for S-IgG and 4.5% for sVNT were inconsistent with the negative results for neutralizing activity of the comparative method.Similar results have been reported in other studies [22].
These findings demonstrate a discrepancy between serological antibody levels and neutralizing activity detected by authentic virus neutralization assay.The binding capacity of virusspecific IgG antibodies is known to increase over time, termed as affinity maturation [23,24].The binding affinity and neutralizing potency of anti-RBD antibodies in SARS-CoV-2 infection have also been reported to increase over time [10,25].This process may be associated with a slower increase in the authentic virus neutralization activity compared to the anti-S IgG and sVNT titers [26].In this study, severe/critical cases of SARS-CoV-2 infection (Group S) showed more rapid evolution of anti-S IgG and sVNT titers than mild/moderate cases (Group M), which was consistent with previous reports [27][28][29].Neutralizing antibodies directly block infection, whereas innate and T-cell responses that mediate neutralizing capacity induce  hyperactivated inflammation and promote severity [30,31].Further studies are required to elucidate whether the rapid induction of anti-S IgG and the persistence of the antibody response contributes to the protection against infection and prevention of severe diseases.
Regarding the role of vaccination and induced humoral immune response after vaccination, we investigated anti-S IgG and sVNT titers in HCWs who received two doses of the BNT162b2 vaccination.A strong correlation between anti-S IgG and sVNT titers was observed in sera approximately 1-2 months after the second vaccination.Significantly higher anti-S IgG and sVNT titers were observed in individuals with a COVID-19 medical history as well as N-IgG positive individuals who have not been diagnosed with COVID-19 due to lack of symptoms.
Of note, no significant difference in both anti-S IgG and sVNT titers were observed in breakthrough infected individuals compared to the ones without infection.Previous studies reported that anti-S IgG and neutralizing antibody titers were inversely related to the increased risk of breakthrough infection [32][33][34].On the other hand, several studies demonstrated no significant difference in antibody titers with or without breakthrough infection [35,36].The discrepancy of the results may have been affected by the measurement time after vaccination, epidemic variants of SARS-CoV-2, immune status of individuals, and sample scale [35].
The production of anti-S IgG or neutralizing antibodies may be related to various factors, such as age, medications, and underlying diseases [37].In our study, B.1.617.2 (delta) was the predominant variant in breakthrough infected individuals.Of the 19 breakthrough infection cases, 13 were diagnosed with a delta variant infection and 6 were unknown in this study.The efficacy of two doses of BNT162b2 vaccine has been reported to be 88.0%against the delta variant [38].
Even if a high antibody titer is obtained through vaccination, breakthrough infection from variants of concern (VOC) will become more likely to occur because the ability to target and neutralize the receptor binding motif (RBM) on the S protein of VOCs is reduced [39,40].Therefore, anti-S IgG and sVNT titers might not be effective indicators of breakthrough infections.
This study has several limitations.Firstly, it was conducted in a single university hospital with a relatively small number of samples.Secondly, there may be an age bias between Group S and Group M of the COVID-19 cases.Finally, post-vaccination antibody measurements were performed only once, and changes in antibody titers over time could not be followed.
In conclusion, using anti-S IgG titers as direct quantitative markers of neutralizing potency is limited.Therefore, serologic tests need to be carefully interpreted in the treatment of COVID-19.On the other hand, anti-S IgG antibody titers can provide information on antibody acquisition both from past infections and vaccination, suggesting that high-throughput, widely available quantitative S-IgG measurements using the Abbott SARS-CoV-2 IgG II Quant assay can be used in epidemiological studies to provide important information for future SARS-CoV-2 infection control.

Fig 2 .Fig 3 .
Fig 2. Longitudinal change of anti-S IgG and sVNT titers.Anti-S IgG titers (A) and sVNT titers (B) of 253 samples from 63 hospitalized patients (Group S, 134 samples from 27 cases; Group M, 119 samples from 36 cases) were plotted against time from symptom onset and fitted (solid line).Red and blue dots indicate the calculated time required to achieve the 50% and the 80% maximal neutralization titer, respectively.The plateau values for anti-S IgG and sVNT titers of Group S and M individuals were shown (Z test).The vertical axes are in logarithmic notation.https://doi.org/10.1371/journal.pone.0291670.g002

Fig 4 .
Fig 4. Anti-S IgG and sVNT titers of the cases with breakthrough infection and non-infected individuals after the second vaccination.Anti-S IgG and sVNT titers in post-second vaccination HCWs (n = 132) were plotted; 19 samples were from individuals with breakthrough infection at a later date (red dots) and 113 samples were from non-infected individuals (black dots).https://doi.org/10.1371/journal.pone.0291670.g004