Vaccine‐induced binding and neutralizing antibodies against Omicron 6 months after a homologous BNT162b2 booster

Abstract Evidence about the long‐term persistence of the booster‐mediated immunity against Omicron is mandatory for pandemic management and deployment of vaccination strategies. A total of 155 healthcare professionals (104 COVID‐19 naive and 51 with a history of SARS‐CoV‐2 infection) received a homologous BNT162b2 booster. Binding antibodies against the spike protein and neutralizing antibodies against Omicron were measured at several time points before and up to 6 months after the booster. Geometric mean titers of measured antibodies were correlated to vaccine efficacy (VE) against symptomatic disease. Compared to the highest response, a significant 10.2‐ and 11.5‐fold decrease in neutralizing titers was observed after 6 months in participants with and without history of SARS‐CoV‐2 infection. A corresponding 2.5‐ and 2.9‐fold decrease in binding antibodies was observed. The estimated T1/2 of neutralizing antibodies in participants with and without history of SARS‐CoV‐2 infection was 42 (95% confidence interval [CI]: 25–137) and 36 days (95% CI: 25–65). Estimated T1/2 were longer for binding antibodies: 168 (95% CI: 116–303) and 139 days (95% CI: 113–180), respectively. Both binding and neutralizing antibodies were strongly correlated to VE (r = 0.83 and 0.89). However, binding and neutralizing antibodies were modestly correlated, and a high proportion of subjects (36.7%) with high binding antibody titers (i.e., >8434 BAU/ml) did not have neutralizing activity. A considerable decay of the humoral response was observed 6 months after the booster, and was strongly correlated with VE. Our study also shows that commercial assays available in clinical laboratories might require adaptation to better predict neutralization in the Omicron era.


| INTRODUCTION
Early efficacy trials and real-world data on the BNT162b2 messenger RNA (mRNA) vaccine confirmed its high effectiveness in reducing laboratory-confirmed infection, COVID-19 hospitalization and death. [1][2][3][4] Nevertheless, a gradual decline in vaccine efficacy (VE) over time has been observed within the first months after the initial two-dose regimen. [4][5][6][7] This waned efficacy was consistent with the decrease of neutralizing antibodies observed by multiple independent studies [8][9][10] supporting neutralizing antibodies as a strong correlate of COVID-19 protection. [11][12][13][14] Moreover, since the beginning of the pandemic, several mutations occurred in the SARS-CoV-2 genome leading the different lineages of the virus. 15 Five of these lineages have been designated as a variant of concern (VOC) by the World Health Organization, namely the Alpha, Beta, Gamma, Delta and Omicron variants. 16 Discovered in November 2021, the Omicron lineage is to date the leading variant over the world. 15 This variant is characterized by 32 dominant mutations in the spike (S) protein, 15 of which are located in the receptor-binding domain (RBD) conferring an increased transmissibility and a considerable immune escape from acquired protection through SARS-CoV-2 vaccination or a previous infection. [17][18][19][20][21][22][23][24] Currently, Omicron is largely dominant and several subvariants have emerged including BA.2, BA.2.12.1, BA.4, and BA.5. 15 All these subvariants have also demonstrated a considerable escape to acquired immunity. 20,25,26 The current BNT162b2 vaccine, which has been elaborated on the sequence of the wild-type SARS-CoV-2, 27 has been shown to be less effective against Omicron compared to other VOCs [28][29][30][31] and the VE also waned over time to reach zero to 22.3% 6 months after the second BNT162b2 dose. 5,7,30,32,33 With the decreased efficacy of vaccines over time and the emergence of highly transmissible SARS-CoV-2 variants that escape neutralization, many countries have deployed third doses of COVID-19 vaccines.
The administration of a homologous BNT162b2 booster dose increased the VE to 58

| Binding antibodies
Binding antibodies against the RBD of the S1 subunit of the SARS-CoV-2 spike protein were measured by the Elecsys Anti-SARS-CoV-2 S assay that measured total antibodies (Roche Diagnostics) with a positivity cut-off of 0.8 BAU/ml. An automatic dilution of 1/100 at >250 BAU/ml was performed by the analyzer to extend the measurement domain up to 25 000 BAU/ml. Additionally, total antibodies against the SARS-CoV-2 NCP (Roche Diagnostics) were measured using the Elecsys Anti-SARS-CoV-2 assay. Results above 0.165 cut-off index were considered positive. 47

| Statistical analyses
Median and IQR were used to present demographic data and geometric mean titers (GMT) and 95% confidence intervals (95% CI) for binding and neutralizing antibodies. The between-group differences were tested using a Tukey multiple comparison test with a multiple testing correction.
The kinetic models for binding and neutralizing antibodies were calculated using the following equation and using nonlog transformed data: Where "a" stands for the maximal antibody response, "b" stands for the baseline response, "c" for the antibody production rate and "d" for the antibody elimination rate.
The elimination rate was obtained from the model which   Figure 3A).

| Binding antibodies
In participants with no history of SARS-  Figure 2). Except at baseline (i.e., just before the administration of the booster), no significant differences were observed in individuals with or without history of SARS-CoV-2 infection (p > 0.05) (  Figure 3B). This threshold represents the binding antibody titer needed to ensure a neutralizing activity of 1/20 ( Figure 4B). Although a waning of binding and neutralizing antibodies was observed as well as similar correlations between mean titers and VE, the two methods used to measure these antibodies were not commutable.

| Binding antibodies versus neutralizing antibodies and correlation to VE
Indeed, neutralizing antibodies, that represent a first layer of adaptive immunity against COVID-19, were only modestly correlated (r = 0.51) against the commercial assay used. This latter was therefore not adapted F I G U R E 5 GMT (±95% CI) of (A) neutralizing antibodies and (B) binding antibodies against the vaccine efficacy against symptomatic disease (%). GMT from individuals with and without previous SARS-CoV-2 infection were merged. Vaccine efficacy (%) were gathered from the literature. Each color corresponds to a single study. CI, confidence interval; GMT, geometric mean titer to predict the presence of neutralizing antibodies. The refining of the cutoff for binding antibodies at 8434 BAU/ml allowed us to improve the prediction of neutralizing antibodies, but the performance remained moderate since there is still a significant proportion of samples with high binding antibody titers that do not correspond to neutralizing activity against Omicron (Figure 3). Therefore, diagnostic companies should need to rethink their current commercial assays (i.e., modification of antigen and epitopes) to design assays capable of predicting neutralizing activity against emerging and highly mutated SARS-CoV-2 variants. 54 This would also avoid any misinterpretation (i.e., high protection in case of high binding antibody titers). 54 Methods used to measure neutralizing antibodies present a low throughput, are time-consuming, need skillful operators, and require high levels of biosafety (especially for live virus neutralization assay). 54 It would therefore be easier to use commercial assays that can be a surrogate of these reference methods.
In our study, we evaluated the vaccine-induced neutralizing activity Vaccine to authorize bivalent formulations of the vaccines. This bivalent vaccine, also called "updated booster," contains two mRNA components of the virus (i.e., one original strain and one in common between the BA.4 and BA.5 lineages). 63 This adapted version is expected to boost the protection against Omicron.

| CONCLUSION
A rapid and significant increase in booster-induced antibodies was observed from 7 days after the homologous BNT162b2 booster.
Thereafter, a considerable antibody waning was noticed within 6 months, which was strongly correlated to VE data available in the literature. The

ACKNOWLEDGMENT
We thank Roche Diagnostics for providing the kits for the evaluation.

CONFLICTS OF INTEREST
The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT
The data presented in this study are available on request from the corresponding author. The data are not publicly available according to the ethical committee decision on the conduct of this study.