key: cord-1016009-ijy1b14n authors: Demirbakan, Hadiye; Koçer, Ipek; Erdoğan, Merve; Bayram, Ayşen title: Assessing humoral immune response after two doses of an inactivated SARS-CoV-2 vaccine (CoronaVac) in healthcare workers date: 2022-01-25 journal: Public Health DOI: 10.1016/j.puhe.2022.01.011 sha: 05f491c6b99af83d6c31d935f92b5068e612b20b doc_id: 1016009 cord_uid: ijy1b14n Objectives During COVID-19 pandemic, the absence of immunity in the population left them susceptible to infection with SARS-CoV-2; healthcare workers (HCWs) being in the highest risk group. This study intends to assess and follow up the humoral immunity in HCWs vaccinated with an inactive virus vaccine (CoronaVac). Study Design This is a prospective observational study. Methods A total of 1072 HCWs were investigated for the presence of IgG antibodies to the receptor binding domain (RBD) of the S1 subunit of the spike protein of SARS-CoV-2 after vaccination. Blood samples were obtained following 28 days of the first dose, 21 days of the second dose, and three months after the second dose. Detection of anti-spike antibodies was performed by the chemiluminescent microparticle immunoassay (CMIA) method (SARS-CoV-2 IgG II Quant, Abbott, Ireland). Results greater than or equal to the cutoff value 50.0 AU/mL were reported as positive. Results Four weeks after the first dose of vaccine, anti-spike antibodies were detected in 834/1072 (77.8%) of HCWs. Seropositivity was higher among females (84.6%) than males (70.6%) (p<0.001) and was found to be highest in both women and men between the ages of 18-34. Anti-spike antibodies were detected in 1008 of 1012 (99.6%) after 21 days of the second dose, and in 803 of 836 (96.1%) after three months of the second dose. Conclusions CoronaVac was found to be highly immunogenic after two consecutive doses performed 28 days apart to HCWs, however the immunogenicity declined significantly (p<0.001) after three months following the second dose of vaccine. Since the Coronavirus disease 2019 (COVID-19) outbreak began, researchers around the world have been trying to develop vaccines against "Severe Acute Respiratory Syndrome Coronavirus 2" (SARS-CoV-2), with more than two hundred vaccines being currently in preclinical or clinical development (1) . Efforts towards the development of a vaccine have led to several candidate vaccines including inactivated vaccines, live virus vaccines, recombinant protein vaccines, vectored vaccines, and DNA or RNA vaccines (2) (3) (4) (5) . CoronaVac is a chemically inactivated whole virus vaccine for COVID-19 developed by Chinese biopharmaceutical company Sinovac Biotech (Beijing, China) and is created from African green monkey kidney cells (Vero cells) that have been inoculated with SARSCoV-2 CN02 strain. It has shown good immunogenicity in mice, rats, and non-human primates with vaccine-induced neutralizing antibodies, which could neutralize ten representative strains of SARS-CoV-2 (6) . CoronaVac was well tolerated and induced humoral responses against SARS-CoV-2, which supported the approval of emergency use of CoronaVac in China in July 2020 (7) . It is being used in vaccination campaigns by certain countries in Asia, South and North America, and Europe also. As of March 2021, 70 million doses of CoronaVac had been administered worldwide. CoronaVac elicited anti-RBD antibodies and neutralizing antibodies in 97.4% of individuals receiving the vaccine at 0 and 28 days (8) . Within the scope of combating COVID-19 pandemic, Turkish Ministry of Health had given emergency use approval (EUA) for the use of CoronaVac and vaccination in Turkey started with priority groups, primarily healthcare workers (HCWs) on 14 January 2021. On 1 June 2021, WHO validated CoronaVac for emergency use, giving countries, funders, procuring agencies and communities the assurance that it meets international standards for safety, efficacy and manufacturing. Vaccination has been shown to provide potent protection from COVID-19, however there are concerns that waning immunity and viral variation may lead to a loss of protection over time (9) . Elucidation of the kinetics and duration of the humoral response induced by active immunization is important for interpreting results from serological surveys and for the management of COVID-19. To determine the humoral immune response induced by CoronaVac against SARS-CoV-2 after two consecutive doses and to guess the need for the administration of a third or booster dose, we planned to detect anti-spike antibodies in HCWs after the first and second doses, as well as after three months following the second dose of J o u r n a l P r e -p r o o f HCWs of both genders, 18 years of age or older, who agreed to participate in this prospective study and those who underwent two-dose (28- The vaccine used in this study was manufactured by Sinovac Biotech (Beijing, China) from inactivated CN02 strain of SARS-CoV-2 created from Vero cells and contained 3 μg/0.5 mL (equivalent to 600 SU per dose) and aluminum hydroxide as adjuvant. Vaccination of HCWs was performed in hospital with the schedule of two consecutive doses of 600 SU (0.5 mL) administered 28 days apart to deltoid. Sequential blood samples were collected from HCWs to determine the levels of anti-spike IgG antibodies; firstly 28 days after the initial dose (between February 11-17, 2021), secondly after 21 days following the second dose (between March 4-10, 2021), and lastly three months after the second dose of vaccination (between May 17-23, 2021). Participants underwent blood sampling with standard venipuncture at the hospital. Transfer of the samples and serum separation were done at the laboratory within 2 hours of collection. Immunoglobulin class G (IgG) antibodies to the RBD of the S1 subunit of the spike protein of SARS-CoV-2 were quantitatively determined from the serum samples. The analysis was performed by the chemiluminescent microparticle immunoassay (CMIA) method using SARS-CoV-2 IgG II Quant kit (Abbott, Ireland) according to the manufacturer's instructions. Detection was carried on with Architect i2000SR instrument (Abbott, Illinois, USA). Test results greater than or equal to the cutoff value stated in assay's package insert (50.0 AU/mL) were reported as reactive and interpreted as positive for SARS-CoV-2 anti-spike IgG antibodies. Results below the cutoff value are reported as non-reactive and interpreted as negative. As descriptive statistics; median and minimum-maximum values for continuous variables, frequency and percentage values for qualitative variables were given. In group comparisons, chi-square test was used. When expected values were less than five Fisher's exact test was used. In all evaluations, p<0.05 was considered statistically significant. Out of 1290 HCWs occupied at the research hospital, 1079 were vaccinated with CoronaVac and 211 refused any vaccination throughout the study period. All vaccinated HCWs were approached for the present study; 7 of them did not want to participate in the study, 1072 volunteers gave written informed consent and completed the two-dose vaccination program. HCWs who refused or were unable to give blood sample after the second dose and/or after three months of the second dose were excluded from the study. The median age of the participants was 33.2 years (95% CI, 0.67: 32.6-33.9 years). The cohort had a slightly greater representation from female individuals, with 51.5% female, 48.5% male. The age distribution of this cohort was as follows: 18 J o u r n a l P r e -p r o o f females (467/552; 84.6%) than males (367/520; 70.6%) (p<0.001) and was found to be highest in both women and men between the ages of 18-34 (88.9% and 79.5%, respectively). Among HCWs between 35-59 years, anti-spike IgG antibodies in females and males were 75.3% and 64.2%, respectively and among those ≥60 years 37.5% in both genders. There was statistically significant difference between all age groups in terms of antibody positivity (p<0.05 for all). Assessment of SARS-CoV-2 anti-spike IgG in HCWs on day 28 after first dose of CoronaVac is given in Table 3 . Of 1072 HCWs 277 (25.8%) informed that they had been previously tested PCR-positive for SARS-CoV-2 on a combined nasal and oropharyngeal swab. Forty-nine (4.6%) of HCWs reported that they were not sure if they had COVID-19 before vaccination, though none had a prior PCR-confirmed diagnosis of COVID-19. The proportion of HCWs infected with SARS-CoV-2 by age group and gender and their anti-spike IgG results are given in Table 3 . Of 1072 HCWs 225 (21%) informed that they had at least one chronic disease; hypertension was the most common reported clinical complaint (59.6%). Only a minority of the participants (2.2%) reported receiving immunosuppressive therapy in the last 12 months. Clinical information of HCWs is given in Table 4 . Participants were required to record any adverse reactions within 28 days after the first dose such as the injection-site adverse events (eg, pain, redness, swelling), headache, or systemic adverse events (eg, fatigue/weakness, fever/chills, muscle/joint pain, and vomiting/diarrhea). Reports indicate that there are over two hundred SARS-CoV-2 vaccine candidates either in development, in initial preclinical stages, or have entered human clinical trials (1). Here we demonstrate the results of a prospective longitudinal study of HCWs to assess the anti-spike IgG positivity after two consecutive doses of an inactivated virus vaccine, CoronaVac. Generally, measurement of the seroprevalence of antibodies, especially neutralizing antibodies, against SARS-CoV-2 from population-based epidemiological surveys is informative for the assessment of the proportion of the population who have at some point been infected with the virus and provides insight into the design of vaccination programs (10, 11) . The reference standard method for detection of neutralizing antibodies, which may be used as a correlate of protective immunity, remains plaque reduction neutralization tests (PRNTs). However, these tests are not routinely performed in clinical laboratories, as they require biosafety level 3 (BSL3) containment facilities, are laborious, and are not amenable to automation (12) . The presence of neutralizing antibodies has been correlated to antibody reactivity to viral structural proteins such as RBD, S, and N using in vitro immunoassays (13, 14) . While data are still limited, there is mounting evidence that antibodies detected by commercial serologic assays correlate with in vitro neutralizing capacity (15) . The sensitivity and specificity of immunoassays were reported to be excellent for detection of the anti-spike humoral response to SARS-CoV-2 infection with a sensitivity between 84-87.1%, specificity between 98.9-100%, and were analogous to the anti-spike antibody assays used during immunogenicity assessments in vaccine clinical trials (16, 17) . Antibodies to spike RBD can inhibit binding of SARS-CoV-2 to angiotensin converting enzyme 2 (ACE2) receptor, generating a strong viral neutralizing response. A wide range of COVID-19 vaccines in development utilize strategies that generate antibody response to the spike protein and the RBD domain of the S1 subunit (18) (19) (20) (21) . Chemiluminescent anti-SARS- CoV-2 serologic assays, as used in this study, have been reported to exhibit high sensitivity (97.8%), as summarized in a systematic review and meta-analysis (22) . In this study we used the Abbott SARS-CoV-2 IgG II Quant kit, which is designed to detect IgG antibodies, including neutralizing antibodies, to the receptor binding domain (RBD) of the S1 subunit of the spike protein of SARS-CoV-2 in serum and plasma. Serum samples obtained from HCWs after the first and second doses of vaccination with CoronaVac showed 77.8% and 99.6% seroconversion, respectively. If we extract HCWs who have had a PCR-confirmed COVID-19 (n=277), or who were not sure to be infected with COVID-19 (n=49) before participating in the study, seropositivity after the first dose remains 70.5% (526/746). Antibody positivity rate was 71.4% (37/49) in HCWs who were not sure whether they had COVID-19 or not. In mRNA vaccine trial studies, anti-spike seroconversion was observed 100% by day 15 following vaccination with mRNA-1273 and by day 21 following vaccination with BNT162b2. (23, 24) . According to our results, CoronaVac reached the seroconversion rate of mRNA vaccines after the second dose (ie. 99.6%) and we found that two doses of this vaccine were highly immunogenic in healthy adults aged 18-59 years. People older than 60 years have an increasing risk of severe illness and death from COVID-19, especially those with underlying chronic conditions. The response to vaccines is usually reduced in older adults due to immune senescence. Zhiwei et al. (25) reported in their phase 1/2 clinical trial that CoronaVac was well tolerated and immunogenic in healthy adults aged 60 years and older, and neutralizing antibody responses to live SARS-CoV-2 were not reduced in that population. Our findings showed that anti-spike antibody response in HCWs ≥ 60 years old (n=24) after the first dose was relatively low (37.5%), however immunogenicity reached a level close to that in the 18-59 age group after the second dose (95.6%). Our study has some limitations; we did not check the seroprevalence of SARS-CoV-2 antispike antibodies in HCWs before vaccination, therefore we could not give data for seroconversion. We mostly reported immune response for healthy adults between 18-59 years of age and included only a small number of individuals from more susceptible groups in our study population (eg, individuals aged ≥60 years or with impaired immunity). Another limitation of this study is; though understanding the duration of the humoral response is essential towards determining immunogenicity obtained with vaccination, antibody testing is not currently recommended to assess immunity after vaccination against SARS-CoV-2. The incidence of adverse reactions was not rare (35.9%), being the most common symptom headache (26.1%). This result was not in accordance with previous finding from another study performed with CoronaVac where the most common symptom was injection-site pain (7) . Compared with other COVID-19 vaccine candidates, such as viral-vectored vaccines or DNA/RNA vaccines, the occurrence of fever (3.2%) with CoronaVac was relatively low (5, 20, 22) . Previous studies suggested that antibodies against SARS-CoV-2 were maintained for at least 4 months (26, 27) . Khoury et al. (9) found that the decay of neutralizing titer in vaccinated subjects over the first 3-4 months after vaccination was at least as rapid as the decay observed in convalescent subjects. However, the SIREN study supported the hypothesis that the new licensed vaccines will provide high degree of immunity of prevention from symptomatic infection with SARS-CoV-2 for working-age adults for an average of 7 months (28) . In this study we observed that the humoral immunity is sustained 96.1% after three months, however the levels of antibody titers obtained in this study should not be used as a correlate of protection, because the protective level of antibody titer was not established to date. As a conclusion, in this study of immunogenicity of an inactivated SARS-CoV-2 vaccine we found that two consecutive doses of CoronaVac was well tolerated with minor adverse reactions and was highly immunogenic in HCWs. As expected, the amount of anti-spike antibodies decreased after three months following the second dose of vaccine and the difference was statistically significant (p<0.001). The antibody level itself might not be the key for an intact immune response, however it is highly predictive of immune protection, and will assist in developing new vaccination strategies to control the pandemic. The durability of humoral responses against SARS-CoV-2 upon vaccination needs to be further clarified with a longer follow-up time. The authors declare that they have no conflicts of interest. World Health Organization. Draft landscape of COVID-19 candidate vaccines SARS-CoV-2 vaccines in development The race for coronavirus vaccines: a graphical guide A strategic approach to COVID-19 vaccine R&D Safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored COVID-19 vaccine: a dose-escalation, openlabel, non-randomised, first-in-human trial Development of an inactivated vaccine candidate for SARS-CoV-2 Safety, tolerability, and immunogenicity of an inactivated SARS-CoV-2 vaccine in healthy adults aged 18-59 years: a randomised, double-blind, placebo-controlled, phase 1/2 clinical trial SARS-CoV-2 immunity: review and applications to phase 3 Neutralizing antibody levels are highly predictive of immune protection from symptomatic SARS-CoV-2 infection Prevalence of SARS-CoV-2 in Spain (ENE-COVID): a nationwide, population-based seroepidemiological study Seroprevalence of immunoglobulin M and G antibodies against SARS-CoV-2 in China The role of antibody testing for SARS-CoV-2: is there one? A comparison of four serological assays for detecting anti-SARS-CoV-2 antibodies in human serum samples from different populations Serological assays estimate highly variable SARS-CoV-2 neutralizing antibody activity in recovered COVID19 patients SARS-CoV-2 seroprevalence and neutralizing activity in donor and patient blood from the San Francisco Bay Area Comparative performance of five commercially available serologic assays to detect antibodies to SARS-CoV-2 and identify individuals with high neutralizing titers Quantitative measurement of anti-SARS-CoV-2 antibodies: analytical and clinical evaluation A COVID-19 vaccine candidate using SpyCatcher multimerization of the SARS-CoV-2 spike protein receptor-binding domain induces potent neutralising antibody responses Phase 1-2 trial of a SARSCoV-2 recombinant spike protein nanoparticle vaccine Two linear epitopes on the SARS-CoV-2 spike protein that elicit neutralising antibodies in COVID-19 patients Safety and immunogenicity of the ChAdOx1 nCoV-19 vaccine against SARS-CoV-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial Diagnostic accuracy of serological tests for Covid-19: systematic review and metaanalysis mRNA-1273 Study Group. An mRNA vaccine against SARS-CoV-2-preliminary report Safety and immunogenicity of two RNA-based Covid-19 vaccine candidates Safety, tolerability, and immunogenicity of an inactivated SARS-CoV-2 vaccine (CoronaVac) in healthy adults aged 60 years and older: a randomised, double-blind, placebo-controlled, phase 1/2 clinical trial Humoral immune response to SARS-CoV-2 in Iceland Robust neutralizing antibodies to SARS-CoV-2 infection persist for months SARS-CoV-2 infection rates of antibody-positive compared with antibody-negative health-care workers in England: a large, multicentre, prospective cohort study (SIREN)