key: cord-0991145-jnbn373w authors: Xie, Hui; Wen, Xiaojing; Li, Juan; Chen, Weixin; Chen, Meng; Zhang, Lichi; Lv, Min; Zhou, Shanshan; Bai, Shuang; Zhao, Wei; Wang, Jian; Wu, Jiang title: Evaluation of Immunogenicity by Pseudovirus Neutralization Assays for Coronavirus Disease 2019 (COVID-19) Variants after Primary and Booster Immunization date: 2022-02-02 journal: Int J Infect Dis DOI: 10.1016/j.ijid.2022.01.068 sha: 91793ae17efb5bb3ffe675e844cb8b3f6ca7fd96 doc_id: 991145 cord_uid: jnbn373w Objectives To determine the status of immune responses after primary and booster immunization for coronavirus disease 2019 (COVID-19) variants and evaluate the differences in disease-resistance based upon titers of neutralizing antibodies (NAbs) against the variants. Methods Participants aged 18 – 59 y received two doses of inactivated COVID-19 vaccine, 14 days apart, and a booster dose after 12 m. Blood samples were collected before vaccination (baseline), 1 and 6 m after primary immunization, and at multiple instances within 21 d of booster dose. NAbs against the spike protein of Wuhan-Hu-1 and three variants were measured using pseudovirus neutralization assays. Results Out of 400 enrolled participants, 387 completed visits scheduled within 6 m of the second dose, and 346 participated received the booster dose in the follow-up research. After 1 m of primary immunization, geometric mean titers (GMTs) of NAbs peaked for Wuhan-Hu-1, while GMTs of other variants were < 30. After 6 m of primary immunization, GMTs of NAbs against all strains were < 30. After 3 d of booster immunization, GMTs were unaltered, seroconversion rates reached approximately 50% after 7 d, and GMTs of NAbs against all strains peaked at 14 d. Conclusion Two-dose of inactivated COVID-19 vaccine induced the formation of NAbs and memory-associated immune responses, and high titers of NAbs against the variants obtained after booster immunization may further improve the effectiveness of the vaccine. The pathogen causing coronavirus disease 2019 has overwhelmed the human immune system and led to severe morbidity and mortality on a global scale. However, scientists all over the world have taken a quick, unprecedented, and coordinated action to develop vaccines and antiviral agents for ending this pandemic. To date, billions of people have been vaccinated with various types of vaccines, including inactivated virus, viral mRNA, and adenovirus vectors. Incidentally, the available evidence demonstrates that all of these vaccines have a commendable expectancy in preventing COVID-19-associated hospitalization and death. In fact, the estimated vaccine effectiveness is 60 -80% in preventing hospitalization and severe disease outcome (Moghadas et al., 2021) and > 80% in preventing death from COVID-19 (Roghani, 2021) . However, with respect to preventing infection, the effectiveness of the vaccines vary at different time points after immunization (Dagan et al., 2021 , Rossman et al., 2021 . This is possibly related to the continuous decline of neutralizing antibodies over time, thereby weakening the effectiveness of the vaccine. In this study, a set of pseudovirus neutralizing antibody assays was established to understand the status of the immune responses at different time points and evaluate the effects of cross immunization against COVID-19 variants after primary and booster immunization. This study was performed between July 2020 and October 2021 in Beijing, China. A total of 400 participants, aged between 18 and 59 y, were recruited. The main exclusion criteria included a history of severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2(SARS-CoV-2), or Middle East respiratory syndrome infection; high-risk epidemiology history within 14 d prior to enrolment (including history of travel or residence in communities with case reports or contact with a SARS-CoV-2-infected individual), axillary temperature > 37.0 °C, and history of allergy to any of the vaccine components. A complete list of exclusion criteria is included in the protocol. Every participant was familiarized with the aim of the study and asked to sign an informed consent agreement. Subsequently, they received two doses of 3 µg of inactivated COVID-19 vaccine (CoronaVac, Sinovac Life Sciences, Beijing, China), 14 d apart according to the product manual. Blood samples were collected from the participants on day 0 (day of first dose vaccine) as well as 1 m and 6 m after the second dose of primary immunization. Out of the 400 participants, 387 completed the visits scheduled within 6 m of the second dose. According to the product manual, the booster immunization can be carried out 6 months after the primary immunization. After 12 m of receiving the second vaccine dose, 346 out of 400 participants took part in the follow-up research, and they were administered a single dose of booster immunization. Thereafter, they were divided into five groups. The participants were included in one of the five groups, according to their wishes. Blood samples were collected separately from 46, 41, 40, 100, and 119 individuals on days 3, 7, 10, 14, and 21 to detect antibodies against the COVID-19 pathogen. Information related to the number of participants and schedule of sample collection is presented in Figure 1 . The study protocol was approved by the Ethics Committee of Beijing CDC (2020-28), and the entire study was performed in accordance with the requirements of Good Clinical Practice of China and the International Conference on Harmonisation. aliquoted (1 mL/tube), and stored at -80 °C for further use. Prior to the pseudovirus titration, a single aliquot of pseudotyped virus was taken out from -80 °C to avoid repeated freezing and thawing. Subsequently, the titration was carried out by serial dilution to infect the target cells Huh-7 in 96-well plates. The 50% tissue culture infectious dose (TCID50) of the pseudotyped virus was calculated according to the Reed-Muench method. The abovementioned procedures were performed according to previously published protocols (Li et al., 2020 , Nie et al., 2020a , Nie et al., 2020b . The titers of neutralizing antibodies were quantified using the pseudovirus neutralization assay for Wuhan-Hu-1 and COVID-19 variants. First, 100 μL of serial 3-fold diluted human serum (starting at 1:10) was incubated with 50 μL of pseudovirus (1300 TCID50/mL) for 1 h at 37 °C in the 96-well plates. Thereafter, Huh-7 cells were added (2 × 10 4 cells/100 μL per well), and the plates were incubated at 37 °C in a humidified atmosphere with 5% CO 2 . Duplicated wells were analyzed for each sample. The cell control (CC), which contained only Huh-7 cells, and the virus control (VC), which contained the virus and the Huh-7 cells, were set up for each plate. After incubation for 24 h, chemiluminescence signals were detected by Molecular Devices SpectraMax® iD5 using the luciferase substrate (PerkinElmer). Reed-Muench method was used to calculate the half-maximal inhibition dilution (ID50), and ID50 values ≥ 30.0 were considered positive. The sample size for this study was based on practical considerations rather than statistical power calculations. Statistical analyses were conducted with GraphPad Prism 8.0.1. Neutralizing antibodies were presented as geometric mean titers (GMTs) with 95% confidence intervals (CIs). The calculations were performed with log10 values of the original data and subsequent application of anti-log transformation. Wilcoxon matched-pairs signed-rank test was used to compare differences among groups. Two-sided p-values < 0.05 were considered statistically significant. There were no detectable titers of neutralizing antibodies against the Wuhan-Hu-1 and three COVID-19 variants (baseline) in the sera of the study participants prior to primary immunization. The seroconversion of the subjects denotes the antibody titer at which their sera convert from negative to positive after 1 m of complete primary vaccination. After 1 m of complete vaccination, the neutralizing antibody titers against Wuhan-Hu-1 increased from baseline to a GMT of 40.2 (95% CI, 36.8 -43.8), and the seroconversion rate was 63.0% (244 of 387 participants). However, the GMTs of the other three variants, B.1.1.7, B.1.351, and B.1.617.2, were < 30, particularly, 29.6 (95% CI, 27.0 -32.4), 10.9 (95% CI, 10.0 -12.0), and 26.1(95% CI, 23.7 -28.7), respectively. There were significant differences among all groups (P < 0.05) ( Figure 2A ). Additionally, their respective seroconversion rates were 53.5% (207 of 387), 13.7% (53 of 387), and 45.7% (177 of 387). Therefore, the GMT and seroconversion rate were the highest for Wuhan-Hu-1 and the lowest for B.1.351. ( Figure 2B ). After 6 m of the second vaccine dose, we observed a rapid decline in the GMTs of neutralizing antibodies against all the COVID-19 pseudovirus strains. In fact, the neutralizing antibody GMTs of all four pseudovirus strains were < 30, namely Wuhan-Hu-1: 13.6 (95% CI, 12.4 -14.9), B.1.1.7: 14.1 (95% CI, 12.8 -15.6), B.1.351: 7.9 (95% CI, 7.3 -8.4), and B.1.617.2: 10.1 (95% CI, 9.3 -11.1). Among them, the GMT of B.1.1.7 was the highest, but there were no significant differences between the neutralization antibody GMTs of B.1.1.7 and Wuhan-Hu-1 (P > 0 05). On the contrary, the GMT of B.1.351, which was the lowest, significantly differed from that of the other groups (P < 0.0001) ( The GMT of the neutralizing antibodies against Wuhan-Hu-1 was 133.2 (95% CI, 114.1 -155.5), and it had increased significantly after booster immunization, as immunization. The seroconversion rates of all the strains were > 90%. Thereafter, the neutralizing antibody titers seemed to reach a plateau phase; apart from the GMT of wild-type, which increased slightly, the GMT levels for all other variants decreased. However, there was no significant difference between the GMTs observed on days 14 and 21 for all the strains (P > 0.05) (Figure 4 ). The present study is a unique large-scale and long-term prospective cohort study on the antibody persistence and secondary immune response of COVID-19 vaccine. The immunogenicities of 387 participants were evaluated prior to vaccination, 1 and 6 m after primary immunization, and at different time points after the booster immunization using pseudovirus neutralization assays for COVID-19. According to previous reports, the peak of neutralizing antibody titers should be seen after 1 m of complete immunization. However, this study demonstrated that after 1 m of primary immunization with inactivated COVID-19 vaccine, there were low levels of antibody response against Wuhan-Hu-1, B.1.1.7, and B.1.617.2 variants, while the neutralization capacity for B.1.351 variant was extremely low. Subsequently, 6 m after the second vaccination dose, there was a rapid decline in the GMTs of neutralizing antibodies, but the antibody positive rates were still approximately 20% for Wuhan-Hu-1 and B.1.1.7 variants. This is consistent with the antibody persistence results of other COVID-19 vaccines that produced high titers of neutralizing antibodies after full inoculation (Doria-Rose et al., 2021 , Favresse et al., 2021 , Pegu et al., 2021 . Incidentally, antibody persistence may be associated with immune memory rather than the titer value of neutralizing antibodies. After 1 y of complete vaccination, 346 participants received 1 dose of booster immunization. To understand the status of the immune response at different time points after the booster immunization, the participants were divided into 5 groups, and blood samples were collected on days 3, 7, 10, 14, and 21. The neutralizing antibody titers did not change in most participants at day 3 after booster immunization. However, based on the approximately 50% seroconversion rate recorded at day 7 d after booster immunization, it may be suggested that for most of the participants, the antibody titers increased significantly between days 5 and 6 of receiving the booster dose. The seroconversion rate reached 90% at day 10, and the GMT levels peaked on day 14 after booster immunization. This sequence of immune response is consistent with the pattern of memory-associated responses, and it further confirmed that a humoral immune response can be induced in the human body by administering inactivated COVID-19 virus vaccines, similar to other vaccines (Chandrashekar et al., 2020) . The most exciting observation is that all variants show excellent immunogenicity after 10 d of booster immunization. This indicates that a high titer of neutralizing antibodies is related to the effectiveness of the vaccine against COVID-19 variant in vitro; however, this is inconsistent with some studies (Caucci et al., 2021) . While moderate levels of antibody titers have been extrapolated to detect infections, extensive real-world data are necessary to support its relation to the protective effects against COVID-19 virus. Hence, it may be suggested that antibody titers were used to indirectly evaluate the effectiveness of the vaccine (McMahan et al., 2021) . This study showed that administration of two vaccine doses could not achieve the expected effect against Wuhan-Hu-1 or the spike protein variants, and a third dose is necessary (Flaxman et al., 2021) . However, the question arises that whether the third dose should be a part of the primary immunization schedule, or should it be included as a booster immunization. Moreover, if the primary immunization requires three doses, then the interval between the second and third doses needs to be determined to obtain best immunogenicity. Pseudovirus neutralization assays can be conveniently used for evaluating vaccine immunogenicity because of their safety, and the effectiveness of this method has been established by a number of studies. Neutralizing antibody titer is the best index to evaluate the immunogenicity of a vaccine (Robbiani et al., 2020) , but it has some limitations (Andualem et al., 2020) . Even though neutralizing antibodies are mainly immunoglobulin (Ig) G, isotype IgA and IgM with neutralization abilities may also be present. Interestingly, the dynamic regularity varies not only among the different classes of antibodies, but also among the different subclasses of one class. In this study, even though the GMT of neutralizing antibodies against Wuhan-Hu-1 was significantly higher (P < 0.0001) than that of B.1.1.7 after 1 m of primary immunization, their immune response levels were similar after 6 m. On the contrary, after 7 d of booster immunization, the GMT of B.1.1.7 antibodies was significantly higher than that of Wuhan-Hu-1 antibodies (P < 0.001). It is unclear whether this is related to the class/subclass of antibodies secreted at different time points, and hence, it needs to be explored further. Some studies have reported that IgG subclasses can interfere with the antibody affinity of COVID-19 virus (Luo et al., 2021 , Stephens and McElrath, 2020 , Suthar et al., 2020 . In fact, this phenomenon has been observed in other viruses too. A study of antibody responses to primary Rubella infection revealed an initial low-avidity IgM response, followed by low-avidity IgG3 and IgA responses, and finally, IgG1 responses maturing from low to high avidity. Therefore, low-avidity antibodies indicate recent infection, and maturation to high avidity antibodies occurs within 2 m post-exanthem (Susan and Stanley, 2018, Wilson et al., 2006) . Future studies can confirm whether the irregular changes in the titers of neutralizing antibodies in some participants indicate a similar pattern in COVID-19 as in the case of Rubella. In conclusion, the neutralizing antibodies induced by two doses of inactivated COVID-19 vaccine can be maintained for 6 m, and high titers of neutralizing antibodies produced after booster immunization can effectively protect against COVID-19 variants in vitro. This work was supported by the Beijing Municipal Science & Technology Commission (Z211100002521014). Hui Xie, Xiaojing Wen, Juan Li, Weixin Chen, Meng Chen, Lichi Zhang, Min Lv, Shanshan Zhou, Shuang Bai, Wei Zhao, Jian Wang, and Jiang Wu all declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Wilson KM, Di Camillo C, Doughty L, Dax EM. Humoral immune response to primary rubella virus infection. 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