key: cord-0259050-lq1ujvpl
authors: Kato, H.; Miyakawa, K.; Ohtake, N.; Go, H.; Yamaoka, Y.; Yajima, S.; Shimada, T.; Goto, A.; Nakajima, H.; Ryo, A.
title: Antibody titers against the Alpha, Beta, Gamma, and Delta variants of SARS-CoV-2 induced by BNT162b2 vaccination measured using automated chemiluminescent enzyme immunoassay
date: 2021-09-26
journal: nan
DOI: 10.1101/2021.09.23.21263927
sha: 6c120ab9820266c4b8c74513effef55b78c3b664
doc_id: 259050
cord_uid: lq1ujvpl

Background Levels of 50% neutralizing titer (NT50) reflect the vaccine-induced humoral immunity after the vaccination against the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Measurements of NT50 are difficult to implement in large quantities. A high-throughput laboratory test is expected for determining the level of herd immunity against SARS-CoV-2. Methods We analyzed samples from 168 Japanese healthcare workers who had completed two doses of the BNT162b2 vaccine. We analyzed immunoglobulin G (IgG) index values against spike protein (SP) using automated chemiluminescent enzyme immunoassay system AIA-CL and analyzed the background factors affecting antibody titer. SP IgG index was compared with 50% neutralization titers. Results The median SP IgG index values of the subjects (mean age = 43 years; 75% female) were 0.1, 1.35, 60.80, and 97.35 before and at 2, 4, and 6 weeks after the first dose, respectively. At 4 and 6 weeks after the first dose, SP IgG titers were found to have positive correlation with NT50 titer (r=0.7535 in 4 weeks; r=0.4376 in 6 weeks). Proportions of the SP IgG index values against the Alpha, Beta, Gamma, and Delta variants compared with the original strain were 2.029, 0.544, 1.017, and 0.6096 respectively. Older age was associated with lower SP IgG titer index 6 weeks after the first dose. Conclusions SP IgG index values were raised at 3 weeks after two doses of BNT162b2 vaccination and have positive correlation with NT50. SP IgG index values were lower in the older individuals and against Beta and Delta strain.

The coronavirus disease 2019 caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global public health threat that has infected 209 million people and caused 4.3 million deaths as of August 19, 2021 . The messenger RNA vaccine gives rapid and robust immunity comparable to natural infection by the virus [1] . The vaccine, which is the cornerstone of current control strategies, was designed to primarily target the SARS-CoV-2 spike protein (SP) of the prototype Wuhan strain [2] . The mRNA vaccines (BNT162b22 and mRNA-1273) elicit high titers of SARS-CoV-2 neutralizing antibodies [3] . Consequently, the efficacy of these vaccines in preventing illness and reducing disease severity range from 94% to 95% [4, 5] . Determining the level of herd immunity against COVID-19 requires mass surveillance of the immune response to SARS-CoV-2 and its variants among vaccinated individuals. Khoury et al. showed that the titers of vaccine-induced neutralizing antibody responses are highly predictive of immune protection [6] . Moreover, measuring the levels of neutralizing antibodies helps to determine the status of an individual's protective immunity against virus infection. Viral neutralization titers measure the ability of antibodies to prevent viral infection of a eukaryotic cell line in vitro. Although the current gold standard neutralization assay requires live SARS-CoV-2, pseudotyped virus-based assays have also been commonly used because of their safety and versatility [7] . However, these conventional neutralization assays are of relatively low throughput and require long readout times. Overcoming these limitations requires the development of alternative means for measuring surrogate antibodies that can replace the cell-mediated neutralization assay. Recently, we developed a dedicated reagents against SARS-CoV-2 spike protein using an automated chemiluminescent enzyme immunoassay (CLEIA) system AIA-CL (TOSOH, Japan). It is a highthroughput serological test capable of processing 120 samples per hour. It simultaneously detects immunoglobulin G (IgG) against the nucleocapsid protein (NP) and SP of SARS-CoV-2 [8] . In tests on COVID-19 patients, an automated chemiluminescent enzyme immunoassay system using the AIA-CL system exhibited 100% sensitivity and specificity in detecting antibodies against SARS-CoV-2. Here, we sought to elucidate the relationship between the level of binding antibodies measured by the CLEIA and neutralization activity determined by a pseudovirus-based neutralization assay in a cohort of donors without a past history of COVID-19. Our aims were, first, to determine the usefulness the commercial CLEIA reagents using the AIA-CL system in assessing vaccine-induced changes in SP-specific IgG (SP IgG) levels against SARS-CoV-2 after BNT162b2 vaccination and comparing these results with the corresponding 50% neutralization titers (NT50); second, to analyze the SP IgG index titers between the original strain and variants of SARS-CoV-2 after BNT162b2 mRNA vaccination using the CLEIA using AIA-CL system designed for the original and these variants; and third, to identify the underlying host factors determining vaccine-elicited humoral immunity, as these factors remain unclear in Asian populations.

. CC-BY 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The hospital has 672 admission beds, including 21 beds for COVID-19 patients. Blood samples were collected from the following four time points: prior to vaccination and then at 2, 4, and 6 weeks after the first dose (the second dose was administered 3 weeks after the first dose). The actual collection day ranged within 3 days before and after the scheduled collection day. The first vaccine dose was administered between March 15 and 22, and the second dose was administered between April 5 and 13. Subjects were asked to provide the following information: birthday and year, sex, drinking habit, smoking habit, comorbidities, body height, and body weight. The participants' age and body mass index at the first dose was calculated. Alcohol drinking habit was categorized corresponding to never drink; occasionally drink or drink several times a week; and drink daily. Smoking habit was categorized corresponding to never smoke, ex-smoker, and current smoker. Comorbidities were defined as having chronic respiratory diseases (including bronchial asthma, diabetes mellitus) and currently undergoing antitumor chemotherapy or immunosuppression, regardless of the doses and types of regimens.

The index value of SARS-CoV-2 SP and NP IgG were measured using the commercial CLEIA reagents (AIA-CL SARS-CoV-2 SP-IgG antibody detection reagent, Tosoh, Japan). Previous validations of these reagents have shown this setting to have high sensitivity and specificity [8] . The supernatants containing the recombinant proteins were collected and purified using the Strep-tag purification system (IBA Lifesciences). Purified these recombinant RBD protein from each variant was immobilized to micromagnetic beads respectively and prepared the prototype reagent panel for each strain on AIA-CL system and their attachment to the AIA-CL instrument have been described previously.

The intensities of the chemiluminescent signals from 100 healthy donor samples were measured with cutoff values (1.0 index value) determined from the mean + 6SD.

The neutralizing assay using an HIV-based pseudovirus bearing the SARS-CoV-2 spike was performed as described previously [9] . Briefly, VeroE6/TMPRSS2 cells were inoculated with pseudoviruses encoding the luciferase reporter gene within mixtures containing five-fold serially diluted serum (1:50 to 1:31250 dilution). Forty-eight hours after inoculation, the luciferase activity of VeroE6/TMPRSS2 . CC-BY 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted September 26, 2021. ; https://doi.org/10.1101/2021.09.23.21263927 doi: medRxiv preprint 5 cells was measured using the GloMax Discover System (Promega). We calculated 50% neutralizing titer (NT50) values using Image J software (NIH). In cases where the serum had no observable neutralizing activity to interpolate NT50, we assigned an NT50 value of 25. All samples were assayed in at least duplicate.

Continuous data are presented either as means with 95% confidence intervals (CIs) or medians with interquartile ranges. Categorical data are presented as numbers and percentages. Continuous variables between two groups were compared using the two-tailed Mann-Whitney U-test, and the Kruskal-Wallis test was used to compare three or more groups. Categorical data were compared using Fisher's exact test. SP IgG and NT50 antibody titers were correlated using Spearman's correlation analysis at 4 and 6 weeks after the first dose. A multivariable regression model was used to investigate the association is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint 

Written informed consent was obtained from the 189 subjects to participate this study. Among them, 168 subjects had completed blood drawn at all time points. Table 1 

We compared SP IgG index values against the original (Wuhan) and three variant strains after vaccination. Our results show that the SP IgG index values binding to the Gamma strain (P.1) were similar to or higher than those of the original strain (Fig. 3) is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint 

With regard to background conditions, there was no significant difference between the SP IgG index values in the subjects according to sex, drinking habit, smoking habit and having any comorbidities ( Table 1 ). The relationship between SP IgG index values and age and body mass index were shown in is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint 

We utilized an automated chemiluminescent enzyme immunoassay (AIA-CL reagent) to investigate the humoral immune response after vaccination against SARS-CoV-2. The AIA-CL method provided good index values of the antibody response after the BNT162b2 vaccination. This method is capable to process a large number of samples rapidly. Our data indicate that SP IgG index values in subjects who had completed two doses of the BNT162b2 vaccine had moderate correlation with neutralizing activities using lentivirus-based pseudovirus assays. This method can be adopted to evaluate subjects that have received different types of COVID-19 vaccines; it can also be used to screen for herd immunity. The BNT162b2 vaccine seemed to induce sufficient immunity at 3 weeks after the second vaccination, not 1 week after the second dose. Our results show that individuals who received only one dose of BNT162b2 did not have sufficient humoral immunity. Currently, the possibility of breakthrough infections after vaccination is raising concerns. According to a case series report, the neutralizing antibody titers in people who developed breakthrough infection were significantly lower than those without breakthrough infection (177.7 vs 501.3) [12] . In this study, a neutralizing antibody index of 100 or an AIA-CL SP IgG index of 10 were observed in the fully vaccinated individuals. However, these indexes should be carefully interpreted and should not be considered as a protective antibody titer.

Higher values of SP IgG index and neutralizing antibody titers are required to lower the risk of developing COVID-19 [11] .

We also detected a difference in the humoral immune responses elicited by BNT162b2 against the original strain and the variants of concern using the prototype reagent panels. Our results demonstrate that the levels of the SP IgG index against the Beta variant were 54% of those of the original strain. By contrast, the SP IgG index value for the Alpha strain was double that of the original strain. Another study reported that the antibody titer against Alpha strain induced by vaccination was not different with that against the original strain [13] . We suggest that the chemiluminescent enzyme immunoassay can capture the difference in humoral responses to the variant strains such as the Beta strain, and this approach can be adopted as a mass screening method.

Several background factors affected the SP IgG index titers in fully vaccinated subjects. We found that older age may be related to a lower antibody response. Regarding age, vaccinated individuals over 80 years old tend to have lower antibody titers [14, 15] . Another report looking at a Japanese population concluded that the antibody titers in the subjects who drink alcohol frequently are lower than those of other cohorts in Japan [16] . The effect of BMI on the antibody titer after vaccination is still controversial. The antibody titer against the SP acquired by natural infection is higher in severely obese patients (BMI > 25) [17] compared to those with BMI <18.5. By contrast, another report suggests that obesity is associated with low antibody titer after vaccination [18, 19] . In our study, drinking habit and BMI did not affect SP IgG index titers. Further research is required to investigate the relationship between individual characteristics and antibody titers induced by BNT162b2 vaccination. Our results . CC-BY 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint Our study had several limitations. First, our study cohort, consisting of 168 subjects, is small.

Cohort size can affect the relationship between predisposing factors and antibody titers. Second, we did not evaluate cell-mediated immunity in our study. SARS-CoV-2 vaccines induce both cell-mediated and humoral immunity [20] . Thus, evaluating cell-mediated immunity contributes to a more thorough analysis of vaccine effectiveness.

Co., Inc. Other authors stated no conflict of interest.

HK contributed to the study design, data collection, statistical analysis and interpretation of data, and the drafting and editing of the manuscript. HG, SY, and TS contributed to the study design and data collection. KM, NO, and YY performed the laboratory tests. TM, AG, HN, and AR contributed to the study design, data collection, and supervised the analysis and preparation of the manuscript. All authors made critical revisions to the manuscript for important intellectual content and approved the final manuscript. All authors meet the ICMJE authorship criteria.

The authors thank all the participants and medical staff for their participation and assistance in the study, is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted September 26, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted September 26, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted September 26, 2021. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted September 26, 2021. ; https://doi.org/10.1101/2021.09.23.21263927 doi: medRxiv preprint Table 1 . Basic characteristics of the subjects, and their SP IgG antibody titers at 3 weeks after the 6 weeks after the first dose. SP IgG index titers were measured using the automated chemiluminescent enzyme immunoassays using AIA-CL system.

Antibodies elicited by mRNA-1273 vaccination bind more broadly to the receptor binding domain than do those from SARS-CoV-2 infection

Evolution of the COVID-19 vaccine development landscape

BNT162b2 induces SARS-CoV-2-neutralising antibodies and T cells in humans

Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine

Impact and effectiveness of mRNA BNT162b2 vaccine against SARS-CoV-2 infections and COVID-19 cases, hospitalisations, and deaths following a nationwide vaccination campaign in Israel: an observational study using national surveillance data

Neutralizing antibody levels are highly predictive of immune protection from symptomatic SARS-CoV-2 infection

A highthroughput neutralizing antibody assay for COVID-19 diagnosis and vaccine evaluation

Development of an Automated Chemiluminescence Assay System for Quantitative Measurement of Multiple Anti-SARS-CoV-2

Months Following Infection in 376 Japanese COVID-19 Survivors

mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants

Immune Correlates Analysis of the mRNA-1273 COVID-19 Vaccine Efficacy

Covid-19 Breakthrough Infections in Vaccinated Health Care Workers

Reduced sensitivity of SARS-CoV-2 variant Delta to antibody neutralization

Age-related immune response heterogeneity to SARS-CoV-2 vaccine BNT162b2

Immune Response to the Biontech/Pfizer BNT162b2 Coronavirus Disease 2019 Vaccination

Antibody responses to BNT162b2 mRNA COVID-19 vaccine and their predictors among healthcare workers in a tertiary referral hospital in Japan

The association between obesity and peak antibody titer response in COVID-19 infection

Central obesity, smoking habit, and hypertension are associated with lower antibody titres in response to COVID-19 mRNA vaccine

Obesity May Hamper SARS-CoV-2 Vaccine Immunogenicities strategies for COVID-19

Early T cell and binding antibody responses are associated with COVID-19 RNA vaccine efficacy onset

Gender Male, N (%)

† SP IgG index titers in the groups were compared using the Mann-Whitney U-test or Kruskal-Wallis test.. CC-BY 4.0 International license It is made available under a perpetuity.is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted September 26, 2021. ; https://doi.org/10.1101/2021.09.23.21263927 doi: medRxiv preprint