key: cord-1028572-dx4ujtz3
authors: Jiang, Ruiwei; Dou, Xiaowen; Li, Min; Wang, Enyun; Hu, Jiwen; Xiong, Dan; Zhang, Xiuming
title: Dynamic observation of SARS‐CoV‐2 IgM, IgG, and neutralizing antibodies in the development of population immunity through COVID‐19 vaccination
date: 2022-03-02
journal: J Clin Lab Anal
DOI: 10.1002/jcla.24325
sha: f0eb960bff16f7f6048e7a72cb06aa4a0424c8d3
doc_id: 1028572
cord_uid: dx4ujtz3

BACKGROUND: Currently, mass vaccine inoculation against coronavirus disease‐2019 (COVID‐19) has been being implemented globally. Rapid and the large‐scale detection of serum neutralizing antibodies (NAbs) laid a foundation for assessing the immune response against SARS‐CoV‐2 infection and vaccine. Additional assessments include the duration of antibodies and the optimal time for a heightened immune response. METHODS: The performance of five surrogate NAbs—three chemiluminescent immunoassay (CLIA) and two enzyme‐linked immunosorbent assays (ELISAs)—and specific IgM and IgG assays were compared using COVID‐19‐vaccinated serum (n = 164). Conventional virus neutralization test (cVNT) was used as a criterion and the diagnostic agreement and correlation of the five assays were evaluated. We studied the antibody responses after the two‐dose vaccine in volunteers up to 6 months. RESULTS: The sensitivity and specificity of five surrogate NAb assays ranged from 84% to 100%. Our cVNT results indicated great consistency with the surrogate assays. At 28 days after primary vaccination, the seropositivities of the NAbs, IgG, and IgM were 6%, 4%, and 13%, respectively. After the booster dose, seropositivities reached 14%, 65%, and 97%, respectively. Six months after receipt of the second dose, the NAb positive rate was eventually maintained at 66%. In all COVID‐19 convalescents, patients were detected with 100% NAb sat three months after discharge. CONCLUSION: COVID‐19 vaccine induced a humoral immune response lasting at least six months. Rapid serological detection was used as a proxy for identifying changes in immunity levels and as a guide to whether an individual may require a booster vaccination.

The global pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been ongoing since 2019, resulting in more than 416 million cases and 5.8 million deaths as of February 17, 2022. 1 Ever-emerging variants like B.1.617.2 that come with a greater ability to disseminate have the chance to prolong the pandemic or even worsen it. The global consensus that developing resistance to coronavirus disease-2019 (COVID-19) is needed has resulted in the acknowledgment that large-scale, global population immunity urgently needed to provide protective immunity and interrupt COVID-19's transmission. [2] [3] [4] Neutralizing antibodies (NAbs) develop from both natural infection and vaccination. Critically, they play major roles in protection against SARS-CoV-2 reinfection, prevention of pneumonia progression, and reduction of overall mortality. 5, 6 Effective vaccination induces B cell responses to produce specific NAbs, which then competitively bind angiotensin-converting enzyme 2 (ACE2) with receptor-binding domain (RBD). Ultimately, this prevents the virus from binding to cellular receptors and entering into cells, thereby preventing infection. NAbs based on S protein detection are an important potential target for the sera of COVID-19 vaccination participants and those who have been exposed to the disease. Previous studies have suggested that the neutralization titer of convalescent serum was approximately the same as that obtained from a vaccinated population. 7 In view of the urgency posed by the COVID-19 pandemic, 143 SARS-CoV-2 vaccines have begun clinical trials, with more than 195 additional vaccine candidates in either pre-clinical or candidate phases. 8 The COVID-19 vaccines included the types of inactivated virus, weakened virus, and mRNA vaccines. 9 61.9% of the world population has received at least one dose of a COVID-19 vaccine. 10 Although vaccine-elicited immunity has been reported in clinical trials, how long Nabs remain responsive in mass vaccination campaigns remains unknown. Given this, there is great need for the development of a rapid strategy for NAb detection in vaccine-protected populations.

NAbs mostly rely on assays using a conventional virus neutralization test (cVNT) and pseudovirus-based virus neutralization test (pVNT). Historically, cVNT has been used as a gold standard for protective NAb assays, but requires strict bio-safety level (BSL-3) facilities and the use of live virus entails an infection risk. Conversely, the alternative pVNT is time-consuming and costly. 11, 12 The critical conditions required for either cVNT or pVNT have greatly limited their use in large-scale Nab screening for the evaluation of population immunity. Surrogate rapid tests have been developed for Nab monitoring, which have included enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), and lateral flow immunoassays (LFAs). 13 Previous work regarding the above has focused on the clinical performance of commercially available SARS-COV-2 serological kits in infectious and recovered COVID-19 individuals; however, long-term monitoring of the immune response in vaccinated cohorts by surrogate NAb assays has remained limited.

Although SARS-COV-2 IgM and IgG are commonly used to diagnose SARS-CoV-2 infection, the specific antibody response in response to the effectiveness of the vaccine remains seldom reported.

In this study, we assessed performance of five surrogate NAb assays and specific antibody tests using cVNT. The characteristics of these assays on the NAb and specific antibodies were then compared. Finally, the dynamics of antibody titers in the vaccinated cohort was monitored using the surrogate assays during a six-month observation period, which sought to evaluate the humoral immune response to the vaccination.

In this long-term study, a total of 164 healthy individuals were from the Shenzhen Luohu People's Hospital between November 2020 and July 2021. Participants received two injections of an inactivated SARS-CoV-2 vaccine (BBIBP-CorV) in the deltoid muscle, with injections being 28 days apart. In total, 46 serum samples were obtained from 10 COVID-19 convalescent patients who had continuous negative RNA results confirmed by reverse transcription-polymerase chain reaction (RT-PCR) tests.

Baseline characteristics of participants are shown in Table S1 .

Briefly, the proportion of male and female participants was 46% and 54%, respectively, with an average age of 42.6 years old (range of 20-85 years old). Among 164 healthy individuals, 59 cases received a six-month follow-up for serological analysis; this included pre-vaccine assessment to six-month post-vaccine. Patients who had recovered from COVID-19 were discharged from the hospital and were visited with a three-month observation after the onset of symptoms. At the time of the first inoculation, blood samples were taken from each volunteer at 0, 14, and 28 days post-vaccination. After a four-week interval, the second vaccination was administered and blood samples were collected after another 7, 14, 28, 90, and 180 days.

Except for the group receiving continuous monitoring, a total of 164 and 122 donors who were 14 days and 28 days, respectively, after completing the vaccination procedure were included for the performance comparison of different antibody assays. Briefly, blood was centrifuged at 3500g for 10 min (Allegra X-15R, Beckman Coulter, USA), and the resulting serum was stored at −80°C prior to all serological tests. The study was authorized by the Ethics Committee of Shenzhen Luohu People's Hospital (2020-LHQRMYY-KYLL-033). All participants gave their written informed consent for participation in this study. The control group without sample was prepared in parallel. Finally, the OD inhibition was calculated by the formula: OD Inhibition (%) = (1-OD sample /OD control ) × 100%. According to the manufacture, the value with OD inhibition above 30% was interpretated as negative result. The operation of Yaneng ELISA kits was similar to cPass, except for the positive cutoff value with OD Inhibition more than 20%.

Conventional VNT test used as a neutralization reference method was performed with 75 samples (14-28 days post-vaccination). The detailed operation was as following. VeroE6 cells were cultivated at 37°C with 5% CO 2 to grow to a monolayer of cells, and the test sera were inactivated at 56°C water bath for 30 min. In the biosafety cabinet, the serum sample was diluted with DMEM containing 2% fetal calf serum at the dilutions of 1:20, 1:40, 1:80, and 1:160, respectively. The equal amount of diluted sera and SARS-CoV-2 strain were incubated at 37°C for 1 h. The virus-sera mixtures were subsequently added to VeroE6 cells in culture plate, the virus titer was about 75 pfu per well. Then the supernatant solution was discarded, DMEM containing 0.8% sodium carboxymethyl cellulose was added and cultured at 37°C with 5% CO 2 for 72 h. When plaque developed, the cells were fixed with 10% formaldehyde solution and stained by 0.5% crystal violet for a plaque-inhibition test. Karber method was applied from the volume of serum and virus at the endpoint of the median tissue culture infectious dose (TCID50), and the titer above 1:20 was identified as NAbs generated in the individuals. cVNT, expressed with ratio and 95% CI. Spearman correlation was used to analyze the correlations of various kits with regard to antibodies titer. The area under the ROC curve (AUC) was estimated to assess the performance of serological assays to detect the presence of IgM, IgG, and NAbs. Wilcoxon rank-sum test was used to determine the diagnostic differences among paired samples, and Cohen Kappa statistic was used to calculate the diagnostic agreement among groups. In view of antibody levels measured in different units by ELISA, CLIA, and cVNT, standardized data processing was carried out with each cutoff value. p < 0.05 was considered statistically significant.

As a standard reference, cVNT was conducted and 50 NAb-positive samples were identified ( Figure 1 ). The sensitivity and specificity were determined with NAb-and NAb-negative samples for the five rapid neutralization assays ( Table 2) . Results indicated that many types of kit were found with good specificity, with a sensitivity ranging from 84% to 100%. The best diagnostic performance was obtained with the Axceed NAb test. The diagnostic consistency (Cohen's Kappa above 0.78) with cVNTs was as follows: Axceed, cPass, iFlash, Ecl, and Yaneng. Using the manufacture's cutoff and NAb titers ≥ 1:20 as our criteria, the AUC of the empirical ROC was assessed for NAb, SARS-CoV-2 IgM, and IgG using the Axceed kits ( Figure S1 ). Diagnostic capacity was obtained for IgM, IgG, and NAb with AUCs of 0.92 (95% CI; 0.86-0.97), 0.99 (0.99-1.00), and 1.00 (1.00-1.00), respectively. The group at 14 days post-vaccine (n = 164) was assessed with IgM, IgG, and five NAb assays. Their resulting seropositivity was 41% for IgM, 88% for IgG, and 92%, 86%, 84%, 82%, and 78% for NAbs using the Axceed, iFlash, Yaneng, cPass, and Ecl tests, respectively.

Using the rapid kit-cPass as a reference, the characteristics of the four surrogate assays were compared. Those that had good consistency with cPass were iFlash and Yaneng (kappa, 0.70), followed by 

The seroconversion for SARS-CoV-2 IgM, IgG, and NAb were obtained from before inoculation to three months after the vaccine using the Axceed assay ( Figure 3A Figure 3G ,F).

At three months and regardless of whether assessing the group with increased or decreased levels, NAb levels were significantly reduced to 6.03 AU/ml when compared with the level observed on day 28

after completing the vaccination procedure. Six months later, the NAb levels were only 25% (the mean from 13.55 AU/ml to 3.34 AU/ ml) of their peak levels.

Surrogate antibody assays were also used in patients who had recovered from COVID-19 and the IgM, IgG, and NAb results are shown in with that of NAb ( Figure 4B,C) . A follow-up study was performed on six convalescent patients ( Figure 4D ), in which SARS-CoV-2 IgM and IgG levels in all six patients had decreased. NAb in recovered individuals were higher than both IgM and IgG; over time, four out of the six patients exhibited a slight decline.

NAb is a key factor in the prevention of SARS-CoV-2, and a neutrali- NAb levels were an important indicator for immunity protection against SARS-CoV-2. 14-16 Both sera produced immunoreactivity, with the predominant antibodies targeted at either RBD or spike protein(S) and which induced a strong neutralization reaction.

Here, the clinical performances of three CLIA assays and two ELISA assays aimed at the RBD protein as well as the specific SARS-CoV-2 IgM and IgG proteins were evaluated and compared with cVNT. We observed good concordance above 0.78 between cVNT and the five surrogate NAb assays. As a surrogate assay, these rapid methods had great specificity at 100% and sensitivity, the latter of which ranged from 84.0% to 100%. It reported 95%-100% sensitivity and 99.93% specificity for cPass kit, which was verified with convalescent serum by cVNT and was ultimately authorized by the in infectious samples at three weeks after symptom onset. 33 Our results showed a good correlation coefficient at 0.69 in convalescents.

Moreover, NAb remained at a long-term positive rate and at a higher titer relative to other antibodies after discharge from hospital. This was likely related to age, sex, disease severity, and/or hospitalization. 34 It was also observed that vaccine-induced NAb levels were lower than those generated from COVID-19 convalescent plasma.

This finding was different from that observed in the study on the KCONVAC vaccine, which showed NAb levels 2.65 times greater than recovered patients. 35 This may be due to a combination of factors, including the great replicability rate of SARS-CoV-2, upregulated expression of encoding proteins, and generation of a strong and robust immune response. The humoral immune responses with sustained NAb titer were essential factors to cellular immunity and also contributed to protecting against SARS-CoV-2 infection and illness and will be required for future study. NAb monitoring in a follow-up cohort was limited to targeting at the RBD protein and may not represent the actual situation (e.g., NAb against unknown or non-RBD proteins). 36 In summary, the surrogate assay was provided as a proxy for the large-scale and dynamic monitoring of NAb titers inCOVID-19 vaccination and infection cohorts. Inactive COVID-19 vaccines generated a gradually emerging humoral immunity response along with the inoculation procedure; critically, antibodies persisted at high titers for at least six months.

None.

The authors declare there are no conflicts of interest to disclose.

All authors were involved in the analysis and interpretation of data as well as drafting the manuscript or revising it critically for 

The data that support the findings of this study are available from the corresponding author upon reasonable request.

https://orcid.org/0000-0002-5668-7281

Min Li https://orcid.org/0000-0002-0624-6381

Xiuming Zhang https://orcid.org/0000-0002-7971-6444

COVID-19) Weekly Epidemiological Update and Weekly Operational Update

The dynamics of humoral immune responses following SARS-CoV-2 infection and the potential for reinfection

Antivirals for COVID-19: a critical review

Antibody-based immunotherapeutics and use of convalescent plasma to counter COVID-19: advances and prospects

Ad26 vaccine protects against SARS-CoV-2 severe clinical disease in hamsters

Protective efficacy of inactivated vaccine against SARS-CoV-2 infection in mice and non-human primates

An mRNA vaccine against SARS-CoV-2-preliminary report

Draft Landscape and Tracker of COVID-19 Candidate Vaccines

Analysis of COVID-19 vaccines: types, thoughts, and application

COVID-19) Vaccinations

A SARS-CoV-2 surrogate virus neutralization test based on antibody-mediated blockage of ACE2-spike protein-protein interaction

Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2

Comparative performance of five commercially available serologic assays to detect antibodies to SARS-CoV-2 and identify individuals with high neutralizing titers

Immunogenicity and safety of a recombinant adenovirus type-5-vectored COVID-19 vaccine in healthy adults aged 18 years or older: a randomised, double-blind, placebo-controlled, phase 2 trial

A systematic review of antibody mediated immunity to coronaviruses: kinetics, correlates of protection, and association with severity

Neutralizing antibodies correlate with protection from SARS-CoV-2 in humans during a fishery vessel outbreak with a high attack rate

Performance evaluation of three automated quantitative immunoassays and their correlation with a surrogate virus neutralization test in coronavirus disease 19 patients and pre-pandemic controls

Structural basis of receptor recognition by SARS-CoV-2

Characteristics of SARS-CoV-2 and COVID-19

SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor

Effectiveness of Covid-19 vaccines against the B.1.617.2 (Delta) variant

Effect of an inactivated vaccine against SARS-CoV-2 on safety and immunogenicity outcomes: interim analysis of 2 randomized clinical trials

Safety and immunogenicity of an inactivated SARS-CoV-2 vaccine, BBV152: a double-blind, randomised, phase 1 trial

Safety and immunogenicity of an inactivated SARS-CoV-2 vaccine, BBIBP-CorV: a randomised, double-blind, placebo-controlled, phase 1/2 trial

Randomized, double-blinded and placebocontrolled phase II trial of an inactivated SARS-CoV-2 vaccine in healthy adults

Neutralizing antibody responses to severe acute respiratory syndrome coronavirus 2 in coronavirus disease 2019 inpatients and convalescent patients

Time course of antibody concentrations against the spike protein of SARS-CoV-2 among healthy hospital workers up to 200 days after their first COVID-19 vaccination

Waning immune humoral response to BNT162b2 Covid-19 vaccine over 6 months

Dynamics of antibody response to BNT162b2 vaccine after six months: a longitudinal prospective study

Immunogenicity of mRNA-1273 COVID vaccine after 6 months surveillance in health care workers; a third dose is necessary

SARS-CoV-2 neutralization with BNT162b2 vaccine dose 3

COVID-19 vaccination and IgG and IgA antibody dynamics in healthcare workers

Longitudinal dynamics of the neutralizing antibody response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection

Sex, age, and hospitalization drive antibody responses in a COVID-19 convalescent plasma donor population

Immunogenicity and safety of a severe acute respiratory syndrome coronavirus 2 inactivated vaccine in healthy adults: randomized, double-blind, and placebo-controlled phase 1 and phase 2 clinical trials

Perspectives on the development of neutralizing antibodies against SARS-CoV-2

Dynamic observation of SARS-CoV-2 IgM, IgG, and neutralizing antibodies in the development of population immunity through COVID-19 vaccination