key: cord-0973939-ztpdesdf
authors: Yildiz, Yesim; Ozger, Hasan Selcuk; Senol, Esin; Karakus, Resul; Buyukkoruk, Merve; Altin, Fatma Betul; Ozcan, Fatma Zehra; Yaman, Melek; Oruklu, Nihan; Yucel, Aysegul Atak; Bagriacik, Emin Umit
title: Evaluation of long term antibody kinetics in healthcare workers (HCWs) vaccinated with inactivated COVID-19 vero cell vaccine (CoronaVac), a propensity score matched observational study Antibody kinetics in HCWs after CoronaVac vaccination
date: 2022-05-11
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2022.05.007
sha: 9b593c404cbbb71fb50b022dbc4a6fc8ed68159a
doc_id: 973939
cord_uid: ztpdesdf

Objectives : This study aimed to evaluate the long-term antibody kinetics after vaccinating with an inactivated COVID-19 vero cell vaccine (CoronaVac) in HCWs of a single center in Turkey. Methods : For this prospective observational study, Chemiluminescence immunoassay (CLIA) and enzyme-linked immunosorbent assay (ELISA) were used for the determination of binding (bAb) and neutralizing antibodies (nAb), respectively. Antibody kinetics were compared for the potantially influencing factors and propensity score analysis was performed to match the subcohort for age. Results : Early bAb and nAb response was achieved in all 343 participants. Titers of bAbs against SARS-CoV-2 on 42 days post-vaccination(dpv) was higher in HCWs who were <40 years of age and who had COVID-19 history. SARS-CoV-2 bAb levels in HCWs on days 42(n=97), 90(n=97), and 180(n=97) were 175 IU/ml (3.9-250), 107 IU/ml (2.4-250) and 66.1 IU/ml (2.57-250), respectively(p<0.001). SARS-CoV-2 bAb(p<0.001) and nAb(p<0.001) titers decreased significantly over time. There was a high negative correlation between SARS-CoV-2 antibody titers and inverse optic density of nAb responses (Pearson correlation coefficient:-0.738, p<0.001). Conclusions : When the antibody responses were compared, it was seen that the vaccine immunogenicity was better in those who had prior COVID-19 history and were <40 years of age. In the course of time, it was determined that there was a significant decrease in bAb and nAb responses after the 90th day. These results may guide approval decisions for booster COVID-19 vaccines.

The Coronavirus Disease 2019 pandemic has affected millions of people worldwide and caused more than five million deaths Dashboard, 2022). Healthcare workers (HCWs) are among the most affected risk groups by the pandemic (Ran et al., 2020; Sim, 2020) . Vaccines play a key role in the control of infection during the pandemic process that the whole world has been in for almost two years (Damasceno et al., 2021) . While many countries all over the world had the opportunity to access mRNA vaccines, inactivated vaccines were the only available option in some countries. Rapidly published mRNA vaccine results were useless in those countries which were implementing inactivated vaccines like Turkey, which started vacccination firstly with HCWs and elderly people against COVID-19 with CoronaVac (Agencies, 2021) .

Coronaviridae. It has four major structural proteins; envelope (E), membrane (M), nucleocapsid (N), and spike (S) protein. Among them S and N proteins are the principal immunogens inducing anti-SARS-CoV-2 specific antibodies (Henss et al., 2021; Walls et al., 2020) . Antibodies that bind to the S protein neutralizes coronaviruses (Henss et al., 2021) .

Neutralizing activity may be detectable nearly up to 14 months after primary infection (Rosati et al., 2021) . S protein is the antigenic target for the development of most vaccines (Sadarangani et al., 2021) . Vaccines stimulate the production of antibodies that inhibit the entry of SARS-CoV-2 into cells. By this mechanism the ACE2-RBD binding interactions and/or S antigen mediated membrane fusion are blocked. Functional neutralizing antibodies (nAbs) are important for viral clearance and protection from re-infection. Vaccineinduced antibody responses, neutralizing activities and duration of these response are different according to type of vaccines (Lim et al., 2021) . Also it is difficult to compare the immunogenicity of COVID-19 vaccines in Phase III clinical trials and further studies, because the lack of standardization in geometric mean titer (GMT) of nAb values, the usage of different immunoassays by different developers, the difference in dosage and administration schedules (Karim, 2021; Rogliani et al., 2021) .

Beijing/China, is a whole-virus inactivated COVID-19 vaccine. The vaccine showed acceptable safety and immunogenicity in healthy adults aged 18-59 and aged 60 years and above in Phase I/II and Phase III clinical trials (Fadlyana et al., 2021; Wu et al., 2021; Zhang et al., 2021) . Turkey started vaccination with CoronaVac on January 13, 2021, with HCWs(COVID-19 Vaccine National Implementation Strategy, 2021). Although inactivated vaccine technologies are familiar with a long history, CoronaVac vaccine has been recently developed for the pandemic. In the limited amount of research conducted on CoronaVac vaccination in healthy adults (Banga Ndzouboukou et al., 2021; Bayram et al., 2021; Bichara et al., 2021; Bueno et al., 2021; Chen et al., 2021; Hunsawong et al., 2021) , maximum 56 days of short-term follow-up was evaluated and better immunogenicity was mostly demonstrated in the participants who had COVID-19 history before vaccination.

The aim of this study was to quantitatively analyse antibodies against SARS-CoV-2 spike protein and to determine the neutralizing activity of the antibodies in the 6-month period following CoronaVac vaccination in HCWs in order to reveal the antibody kinetics.

This prospective observational study was conducted on HCWs at the Gazi University Hospital 

The inclusion criteria for the study were age over 18 years and getting two shots of Coronavac 28 days apart. The exclusion criteria were; confirmed SARS-CoV-2 PCR positivity, after the first dose of Coronavac vaccine and having any condition that has a known affect on vaccine response like pregnancy and immunosuppressive condition or drug usage.

Prior COVID-19 history is defined as the diagnose of COVID-19 confirmed with SARS-CoV-2 PCR positivity, before the first dose of CoronaVac vaccine. Those who had positive SARS-CoV-2 PCR test result in the follow-up after the first dose of vaccine were excluded.

The results of SARS-CoV-2 PCR tests, taken from HCWs who had symptoms or were in contact with the symptomatic case were obtained from the hospital surveillance system.

The antibody titer change (Δ) is calculated as the difference between the GMT values of the binding antibody (bAb) titers detected by ELISA between days 42-90 and 42-180.

In this study, we aimed to evaluate the antibody kinetics on the 42nd, 90th and 180th day after the first dose of CoronaVac, without interfering with the vaccination decision of the volunteers. Sera (3-4 ml of venous blood sample) were obtained on the 42nd, 90th and 180th days after the first dose of vaccination, respectively. All specimens were coded prior to processing. Flow-chart of the study is shown in Fig 1 The demographic information and medical history of the volunteers (comorbidity, drug usage, allergy history, SARS-CoV-2 PCR positivity date, first and second dose vaccination dates) were recorded via questionnaire at the time of taking initial serum samples. The blood samples taken were centrifuged at 3000 rpm for 10 minutes in Gazi University Hospital Infectious Diseases laboratory, and the separated sera were taken into 2 ml Eppendorf tubes and stored at -86 degrees. On the study day, serum samples removed from the freezer and transferred to Gazi University Department of Immunology Laboratory, accordingly with the relevant guidelines(CDC, 2020; COVID-19 (SARS-CoV-2 INFECTION) LABORATORY BIOSAFETY GUIDE, 2020).

All serum samples were analyzed with a chemiluminescence immunoassay (Roche, Elecsys®

Anti-SARS-CoV-2 S Quant) developed for quantitative determination of antibodies against SARS-CoV-2 spike protein on Cobas 8000 e801 system available in the Gazi University Diagnostic Immunology Laboratory. According to the manufacturers instructions, a titer of ≥ 0.8 U/mL is considered reactive, and a titer of < 0.8 U/mL is considered non-reactive.

All serum samples were analyzed with ELISA for determination of inhibitory activity of RBD-ACE2 binding induced by nAbs to SARS-CoV-2 in human sera. of neutralizing antibodies is reflected by more lower signal formation. Calculations were made according to the formula as stated in the user manual of the test. Accordingly, the average of the negative controls (NC) was divided into 2 and the cut off value (mean NC/2=cut-off (Co)) was calculated. This value was used to interpretation of the results. For interpretation, the ratio of the Co and sample OD450nm/620-630nm (S) were found. If the Co/S value is less than and equal to 1, it is considered as negative and if it is greater than 1, it is considered as positive result.

All data were analyzed by IBM SPSS Statistics for Mac, version 25.0 for Mac OS X (IBM Corp., Armonk, N.Y., USA). The normality of the data distribution was determined by the Shapiro-Wilk test, histogram, and Q-Q plots. The categorical values of the patients were expressed as a number and a percentage and were analyzed with a Chi-square test. Continued values were presented as a mean and standard deviation (SD) or median values and an interquartile range (IQR) of 25%-75%. The non-parametric values were analyzed using the Mann-Whitney U, and the parametric ones with a Student t-test. One-way anova test was used to compare the geometric mean of SARS-CoV-2 antibodies by risk factors. Comparison of non-parametric and parameteric dependent variables was done with Friedman test and Cochrane Q test. Vaccine responses were compared in the cohort for potential factors that could influence the antibody response and in the subcohort matched for age with propensity matching. The 95% confidence intervals (95% CIs) were calculated whenever appropriate, and a two-tailed p-value <0.05 was considered statistically significant. Graphs were created using the GraphPad version 9 program.

HCWs were enrolled in the study. The median age of the participants was 38 (29-47) and 63.6% (n=218) of them were female. 37.5% of HCWs (n=112) had COVID-19 history prior to vaccination. Of these 112 HCWs, 35.3% (n=40) had COVID-19 in the last 3 months, 39.2% (n=44) in between 3-6 months and 25% (n=28) more than 6 months before vaccination.

At day 42, the GMT of binding antibodies against spike protein of SARS-CoV-2 after two doses of CoronaVac vaccine was 168 (3.9-250).

The SARS-CoV-2 antibody response of the HCWs on 42 dpv was evaluated according to age, sex and COVID-19 history (Table 1) . Comparison of GMTs of SARS-CoV-2 bAbs and percentage of ACE2-RBD nAbs responses in 97 HCWs for age and history of COVID-19 on 42, 90 and 180 dpv is evaluated in Table 2 and Vaccine responses for age and COVID-19 history were compared in the study cohort and in the age-matched subcohort (Table 3) . There was a negative high correlation between bAb titers against SARS-CoV-2 and ODs of nAb responses (Pearson correlation coefficient:-0.738, p<0.001). The correlation between bAb titers against SARS-CoV-2 and ODs of nAb responses was evaluated according to COVID-19 history and age (Fig 4 and Fig 5) . In current literature early immune response for CoronaVac was evaluated both in phase trials that found CoronoVac was correlated with high early seroconversion rates in healthy adults (Bayram et al., 2021; Bueno et al., 2021; Dinc et al., 2022; Fadlyana et al., 2021; Uysal et al., 2021) . Similarly we identified this high early antibody response, 42 days after the first shot of CoronaVac. According to recent studies, the main factors that could influence the vaccine response are young age, female gender, and COVID-19 history before vaccination (Banga Ndzouboukou et al., 2021; Bayram et al., 2021; Dinc et al., 2022; Şenol Akar et al., 2021; Soysal et al., 2021; Uysal et al., 2021) . Vaccine immunogenicity is affected by age, in previous studies. Although high immunogenicity was demonstrated in phase I-II trials of CoronaVac in the elderly (Wu et al., 2021) , this was neither found in our study nor other studies in the literature (Karamese & Tutuncu, 2022 the frequency of anti-SARS-CoV-2 nAbs was higher in people younger than 40 years of age but significantly decreased with advancing age, supporting our findings (Bichara et al., 2021) .

Also, in some studies, COVID-19 history prior to vaccination was associated with higher early antibody response (21-28 days) after CoronaVac vaccination (Bayram et al., 2021; Dinc et al., 2022; Soysal et al., 2021) . In our study, the frequency of previous history of COVID-19 was higher in the younger group. Therefore, when the comparison groups were matched for these risk factors with propensity matching, age was identified as the main factor influencing early antibody responses in our study.

Long-term antibody responses are still uncertain after CoronaVac vaccination, as most of the studies evaluate the short-term nAb responses (Bichara et al., 2021; Chen et al., 2021; Şenol Akar et al., 2021; Soysal et al., 2021) . When the CoronaVac studies are evaluated, the longest follow-up period is 8 weeks after the second dose of vaccination. It is stated that the bAb and nAb responses obtained in these studies remained stable during the follow-up periods. Unlike these studies, we as well evaluated long-term antibody response for up to 6 months at 42, 90, and 180 dpv in our study. However, high bAb and nAb responses on 42 dpv, significantly decreased after 90 days and nAb responses were found 80.4% at 180 dpv. The bAb and nAb responses decreased significantly at 180th day, especially in HCWs over 40 years of age and without a history of COVID-19. This result suggests that

CoronaVac-associated antibody response should not be relied upon after 90 days mainly in these groups.

Our main limitation is that the study was conducted only in a certain occupational group therefore the results of our study cannot be generalized to the whole population. The positivity of IgG antibody to SARS-CoV-2 at baseline before vaccine was not evaluated in participants. In this study, only the humoral (neutralizing antibody) response of the vaccine was revealed, no information on cellular immunity was presented. That's why, our results cannot be considered to provide sufficient evidence as to the extent to which this vaccine will protect individuals from the disease.

In conclusion, on 42 dpv, the majority of HCWs developed a significiant early antibody response following the administration of two doses of CoronaVac 28 days apart.

Binding and neutralizing antibody responses diminished over time especially after 90 dpv and especially in HCWs who did not previously infected with SARS-CoV-2 and who were >40 years of age. According to our findings, booster vaccine doses should be planned in terms of age and previous COVID-19 history. These results will help to define immunogenicity and protection of CoronaVac in HCWs who are struggling at the forefront of the pandemic, and may guide further approval decisions for booster strategies in COVID-19 vaccination implementations.

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We thank the study participants for their generosity and continued support of COVID-19 vaccine research. Our abstract (abstract number 03919) has been accepted as poster presentation in 32nd European Congress of Clinical Microbiology and Infectious Diseases (ECCMID) 2022.

No potential conflict of interest was reported by the author(s).

Coordination Unit (Grant number TGA-2021-7097).