key: cord-0920620-7b26zl89
authors: Oliveira-Silva, Joana; Reis, Teresa; Lopes, Cristiana; Batista-Silva, Ricardo; Ribeiro, Ricardo; Marques, Gilberto; Pacheco, Vania; Rodrigues, Tiago; Afonso, Alexandre; Pinheiro, Vítor; Araújo, Lucília; Rodrigues, Fernando; Antunes, Isabel
title: Long term serological SARS-CoV-2 IgG kinetics following mRNA COVID-19 vaccine: real-world data from a large cohort of healthcare workers
date: 2022-05-11
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2022.05.026
sha: 41ecace250de877607679b95fefa490b31a7082c
doc_id: 920620
cord_uid: 7b26zl89

Objectives : To assess kinetics and predictive variables of humoral immune response to mRNA SARS-CoV-2 vaccine administration. Methods : Blood samples were collected before (T0), and 15-, 90- and 180-days following vaccination (T1, T2 and T3, respectively). The Quant SARS-CoV-2 IgG II Chemiluminescent Microparticle Immunoassay (CMIA) was used to determine anti-spike IgG. Results : Almost 3000 healthcare collected blood samples in the three time points: fifteen days post-vaccination 97.6% subjects presented a robust IgG anti-spike response (>4160 AU/mL); than at three and six months it decreased in median 6.5-fold to 35.0% and 3.0-fold to 3.3%, respectively. Linear mixed effects model supported that female gender, younger age groups and seropositive pre-vaccination maintained higher antibody titers. Curves became tighter with time progression, although titers from seroposive subjects decrease at a slower rate than seronegative ones. Conclusion : These findings strengthen the case for steeply decrease of anti-SARS-CoV-2 antibodies up to 6-months, suggesting that serological evaluation might guide the need for periodic boosters in specific groups prone to lower antibody titers.

In late 2019, SARS-CoV-2 triggered a new pandemic. Vaccines started, urgently, to be developed and U.S. Food and Drug Administration (FDA) authorized their use in an emergence context on the 11 th of December of 2020 after demonstrating 95% efficacy (Food and Drug Administration, 2020) . In Portugal, healthcare workers (HCW) received the first doses of BNT 162b2 mRNA COVID-19 vaccine (Pfizer/BioNTech) by the end of December 2021. Vaccine efficacy against Covid-19 was 91.3% through 6 months of follow-up in subjects without evidence of previous SARS-CoV-2, thus reflecting a gradual decline in vaccine efficacy (Stephen J. Thomas et al., 2021) .

By this time, numerous studies of IgG humoral immunity started to be developed to better understand the kinetics of antibodies (Oliveira-silva et al., 2021; Salvagno, Henry, Pighi, De Nitto, et al., 2021; Sasso et al., 2021; Tré-Hardy et al., 2021) . Nevertheless, the long-term duration of humoral immunity from SARS-CoV-2 vaccine remains unclear, due to lack of data from large, real-world studies. Bayart et al. (Bayart et al., 2021) , observed waning of IgG antibodies over time, thought at 180 days after vaccination, subjects still had detectable anti-Spike antibodies. As reported elsewhere, after the first contact with the virus, B cells produce antibodies that decrease over months, particularly in elderly, men and immunosuppressed subjects (Chavarot et al., 2021; Geisen et al., 2021; Levin et al., 2021) . Concordantly, also previously infected individuals, maintained higher IgG titers over three month studies (Tré-Hardy et al., 2021) , (Lau et al., 2021) .

Considering that antibody titers might be a good biomarker for the protective efficacy of antibodies and successful humoral immune responses after SARS-CoV-2 exposure or vaccination, it is considered that SARS-CoV-2 IgG kinetics concedes relevant information concerning immune status and a proxy for immunization status (Bayart et al., 2021; Levin et al., 2021) . In this study, we report humoral immunity data of the first 6 months follow up postvaccination from a large cohort, which emphasizes the decline of IgG antibodies.

Healthcare workers (HCW) from the Centro Hospitalar e Universitário de Coimbra (CHUC) were vaccinated in late December 2020 with BNT162b2 mRNA and included in a prospective cohort to evaluate SARS-CoV-2 IgG serological kinetics. Subjects were tested for anti-spike IgG antibody before the first dose (T0), and then 15 days (T1), 3 months (T2) and 6 months (T3) after completion of the second dose. HCW with prior diagnosis of SARS-CoV-2 were excluded from the first phase of vaccination. Only subjects with complete serological data at all timepoints, were included in analyses (n=2968). In this population most HCW were naïve (seronegative for SARS-CoV-2 IgG before vaccination) while there was sixty-three seropositive (due to eventual asymptomatic past contact with the virus). This study was approved by hospital's Ethics Committee (OBS.SF.106-2021) and deferred consent was obtained under stringent application of ethical and legal procedures for data collection, such as protection of confidentiality of the personal data and mitigation of risks to privacy.

Blood was collected from each participant at every timepoint and processed to serum within 4 hours. A chemiluminescent microparticle immunoassay (CMIA) SARS-CoV-2 IgG II Quant was used to determine IgG anti-spike, receptor-binding domain (RBD) S1 subunit of SARS-CoV-2 on Alinity i (Abbott Laboratories). The cut-off and upper detection limit of Abbott S-RBD IgG test were 50 and 80 000 AU/mL, respectively, whereas sensitivity and specificity were 99.37% and 99.55%. As per manufacturer recommendations, antibody titers above 50 AU/mL were considered reactive. We used IgG antibody titers > 4160 AU/mL as an indicator of strong neutralizing activity, as previously reported (Ebinger et al., 2021) . All measurements were undertaken following appropriate quality control procedures, daily performed for routine clinical assessment of SARS-CoV-2 IgG.

Departure from normality was tested using Shapiro-Wilk test, and data presented as median (M) and interquartile range (IQR). For longitudinal comparison of SARS-CoV-2 IgG titers between timepoints (T0, T1, T2 and T3), the Friedman's followed by Wilcoxon tests were used, with

Bonferroni correction for multicomparison. To assess differences among independent variables (gender, age groups and reactive titers in T0) at each timepoint, the Mann-Whitney or Kruskal-Wallis tests were applied.

We modelled the decrease after vaccination (over T1, T2 and T3) using a linear regression model with mixed effects. Our data was grouped by subject. This model is appropriate for longitudinal data and extends the linear model adding random effects that can be seen in terms of additional error, accommodating correlation between observations from the same individual. Fixed effect covariates included gender, age group (18-30, 30-40, 40-50, 50-60 and >60 years) , humoral status prior to vaccination (T0 above or below 50 AU/ml), and time (in months). Interactions with time were also included. After log10 transformation of IgG titers, models were fitted with population-level fixed effects and individual-level random effects (Worker ID), for intercept and slope. Models with random effects only for intercept were also fitted. We started by fitting the null model, only including the outcome variable and individual-level random effects. The model presented is the model that fitted our data better. Models' comparison was conducted using the difference in Akaike information criterion (AIC) above 4 (Burnham et al, 2011 ) as significant and fitted using maximum likelihood. We estimated the marginalized R 2 , the proportion of variance explained by the fixed effects (Nakagawa and Schielzeth, 2013) , and the conditional R 2 of the model that is the proportion of variance explained by both the fixed and random factors (Nakagawa and Schielzeth, 2013) . Statistical analyses were conducted using R (version 4.0.05) and linear model with mixed effects was fitted using the lmer function (lme4 package).

Data was collected between December of 2020 until August of 2021. Close to three thousand subjects participated in this study, with median age of 45 years (IQR 36-to-55) (77.5% females).

All had full data on IgG titers at the timepoints T0-T1-T2 and T3. Before vaccination, most participants were naive (median=6.8, IQR= 6.8-6.8 AU/mL), although 2.1% (n=63) of the subjects had IgG anti-SARS-CoV-2 above cut-off (>50 AU/mL) but below 4160 AU/mL. Post vaccination, test reactivity (>50 AU/mL) was maintained throughout the study in 99.9, 99.8 and 99.7% of the population, at T1, T2 and T3 timepoints, respectively. Fifteen days postvaccination (median IgG=21.3x10 3 , IQR=13.3x10 3 -33.0x10 3 AU/mL), 97.6% subjects presented a robust humoral response (>4160 AU/mL), whereas at three months (median=3.2x10 3 , IQR= 2.0x10 3 -5.1 x10 3 AU/mL) it decreased in median 6.5-fold to 35.0% and then by 3.0-fold to 3.3% at 6 months (median=1.0x10 3 , IQR= 0.64x10 3 -1.6x10 3 AU/mL). The Friedman's test [ 2 (3)=8652.4, P<0.0001] revealed a statistically significant difference in SARS-CoV-2 IgG throughout the follow up, further confirmed by Willcoxon between timepoints (P<0.0001) ( Figure 1 ).The comparison between strata of the independent variables, gender, age group and IgG reactivity before vaccination (Table 1) .

Regarding the mixed effects model, the final model included random effects for the intercept and slope. The marginalized and the conditional R 2 were 0.71 and 0.91, respectively.

Female gender, previous reactive titers and younger age group, each contributed to higher antibody levels at the first timepoint following vaccination (Table 2 ). We verified, that every month, the log-transformed IgG levels decreased 0.230 times (p<0.001). Interaction of gender and age with time were strongly correlated with the variate time and were excluded. (Table 3 ).

The variable interaction of time and IgG titers for seropositive participants was significant (P=0.0002). Therefore, antibody levels from participants seropositive at T0 showed higher values after vaccination and decrese at a slower rate (-0.23 vs -0.168, p<0.001) suggesting that at 6 months post-vaccination the IgG levels remain divergent.

This real-world study of COVID-19 humoral response following BNT162b2 vaccination, demonstrated a significant decline in anti-spike IgG titers 6 months post vaccination. Despite an early increase at 15 days after completing the second dose, the IgG levels decreased significantly at both 3-and 6-month timepoints. Our findings, agree with data reported by others (Gaebler et al., 2021; Naaber et al., 2021; Salvagno, Henry, Pighi, Nitto, et al., 2021) .

A recently published randomized placebo-controlled clinical trial, following up over forty thousand subjects vaccinated with BNT162b2 for COVID-19, described that effectiveness peaked at 96.2% during the first 2 months after second dose, and declining to 83.7% in the 4-6 months post-immunization, marking an average decline of 6% every two months (Stephen J. Thomas et al., 2021; Tartof et al., 2021) .

After vaccination with the BNT162b2 vaccine, anti-SARS-CoV-2 IgG kinetics tend to peak around 4 to 30 days, followed by a substantial reduction over time, with significantly lower levels at 6-months (Levin et al., 2021) , (Naaber et al., 2021) . Here, in a large cohort of HCW, we observed that although post-vaccination IgG titers was reactive (>50 AU/mL) for over 99.5% of the population at T1, T2 and T3 timepoints, when we used the cut off indicating a robust humoral response (>4160 AU/mL), the frequency of participants declined by 6.5-fold from 97.6% after 15 days to 35.0% at 3-months, and then by 3.0-fold to 3.3% at 6-months. Similar studies with reduced number of participants yielded common findings (Bayart et al., 2021; Levin et al., 2021) . Seemingly, the decrease in IgG levels throughout post-vaccination follow up occurs in parallel with neutralization titers (Terpos et al., 2021) . In our study, the significant decrease in titers was independent of gender, age or IgG reactivity before vaccination, which is in agreement with previous data (Dan et al., 2021) . The lack of proportionality between the decline in mRNA vaccine effectiveness and the waning humoral immune response kinetics over time, suggests that during post-vaccination follow up the protection might become dependent from immunological mechanisms other than humoral. Noteworthy, declines in effectiveness of COVID-19 vaccine have also been attributed to the widespread dissemination of the delta variant (Bayart et al., 2021) .

The efficacy of humoral immunity alone, against SARS-CoV-2, has been questioned and the relevance of T cell memory evaluated. Studies investigating antibody and T cell responses in matched samples of convalescent patients revealed decreasing spike-specific and stable nucleocapsid-specific antibody responses, whereas functional T-cell responses remained robust, increasing in both frequency and intensity (Bilich et al., 2021) . Circulating antibody titers were shown to be not predictive of T cell response to SARS-CoV-2 (Dan et al., 2021) . Noteworthy, while IgG antibodies decreased significantly over time, the number of RBD-specific memory B cells remained unchanged at 6 months after infection (Gaebler et al., 2021) . In fact, despite a slight decrease in association with age, memory B cells seem to be efficiently primed by mRNA vaccination and detectable after the second vaccine dose, which concedes memory B cells a role in mounting recall responses to SARS-CoV-2 (Goel et al., 2021) .

Taken together, our and other's findings suggest that serological tests for SARS-CoV-2 might not reflect the immune memory response in terms of robustness and durability, highlighting the need to determine cellular responses in addition to serologies (Cromer et al., 2021; Tretyn et al., 2021) .

We found significant differences in antibody titers between naïve versus seropositive subjects before vaccination, and in each of the subsequent timepoints. There was a trend to decrease in absolute difference as time to depart from antibody peak. Those with reactive titers prior to vaccination remained with higher levels at 6-month follow up. Indeed, the mixed effects linear model revealed that being seropositive at T0 contributes for higher antibody levels, at the peak and during the observed period. These results agree with previous studies showing that baseline seropositives have longer estimated half-life and less accentuated decline in SARS-CoV-2 IgG titers (Bayart et al., 2021; Salvagno, Henry, Pighi, Nitto, et al., 2021 , Zhong, et al., 2021 .

Age has been inversely related with immune response in all timepoints of the post-vaccination follow up. We observed that median IgG antibody levels decreased over a six-month period in all age groups, although the difference among age groups decreased over time. Nonetheless, in the mixed-effects model, age remained as a significant independent factor to predict antibody levels.

These results are in line with other reports, that observed a negative correlation between age and antibody levels (Naaber et al., 2021; Salvagno, Henry, Pighi, Nitto, et al., 2021) , and with neutralizing antibodies (Salvagno, Henry, Pighi, Nitto, et al., 2021) . Given that our study is from a working population, subjects over 68 years old were not included. Nonetheless, evidence suggests there is a lower humoral response at 6 month after the vaccine for patients above 60 (Tretyn et al., 2021) and over 65 years old (Levin et al., 2021) .

Gender remained a significant factor throughout all timepoints analysed, with female health care workers presenting higher titers than male counterparts, following vaccination. The linear model with mixed effects showed that female gender contributed independently towards higher antibody levels, albeit this effect seems to decrease trough time. These observations agree with previous reports (Levin et al., 2021; Salvagno, Henry, Pighi, Nitto, et al., 2021) .

This study included a cohort of healthcare workers, a professional group exposed to occupational risk to COVID-19. Eligible participants were active workers, younger than 67 years of age, without substantial comorbidities, with only limited generalizability to elderly and to adults with serious comorbidities. Although initially designed to include only naïve subjects, a small number of participants were found to be seropositive. Therefore, findings comparing titers from naïve versus seropositive participants should be interpreted cautiously, despite they are in line with other larger studies (Bayart et al., 2021; Ebinger et al., 2021) . Here, we focused on serological evaluation of immunological response to COVID-19 mRNA vaccination, even though the immune response to the vaccine is multifaceted and involve neutralizing antibodies and T memory cells, beyond IgG antibodies, in the post-vaccination protection (Krause et al., 2021) .

Notwithstanding those limitations, we present serological data from a cohort with a large sample size and a longer follow-up period compared to others in the literature. Moreover, we used a mixed-effects model, suitable for longitudinal datasets where multiple correlated measurements were taken from each subject, allowing more accurate and precise estimates of population heterogeneity (Bottino et al., 2021) .

Data presented here provides further evidence for the eventual requirement of a SARS-CoV-2 IgG serology-guided booster vaccinations. Despite controversial, this strategy has been adopted by some countries for elderly subjects and immunocompromised patients with over 6-months post-vaccination follow up time ( Bar-On et al., 2021; Krause et al., 2021) .

Albeit we present data of IgG antibodies decline over time, which could be expected, provided that not all vaccine-induced plasmablasts commit or are maintained as long-lived memory plasma cells (Naaber et al., 2021) , it is also well established that vaccine efficacy remains high after six months (S. J. Thomas et al., 2021) , so even if humoral immunity appears to wane, it does not necessarily mean a reduction in efficacy (Krause et al., no date) .

The decline of specific anti-SARS-CoV-2 IgG antibodies over time through 6-months postvaccination suggests waning of humoral immunity and impaired capacity to fight the virus and supports the need to re-activate IgG production. This is a cohort study planned for a one year follow up, which will permit sharpen the antibodies' kinetics model. Accurately evaluated antibody response, together with cellular immunity status, and other covariates including age and gender, may add to clinical reasoning to support the individualization of the immunization plan.

This work further contributes to delineate the pattern of the immune response to COVID-19 mRNA vaccine, fostering additional research to determine the titers needed for protection. Table 1 . Serological levels SARS-CoV-2 IgG, overall and by strata of gender, age group and IgG reactivity, before and following COVID-19 mRNA vaccination. Data are presented as median and interquartile range. 

LA, FR, IA contributed to conceptualization, supervision, and to final review and editing of the manuscript

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** Kruskal-Wallis test, followed by Mann-Whitney tests. a P<0.0001 for all comparisons except 40-50 vs 50-60 (P=0.288), 40-50 vs >60 (P=0.002) and 50-60 vs >60 (P=0.027) (Mann-Whitney tests). b P<0.0001 for all comparisons except