Article Figures and data Abstract Editor's evaluation Introduction Materials and methods Results Discussion Data availability References Decision letter Author response Article and author information Metrics Abstract Background: The development of vaccines to control the coronavirus disease 2019 (COVID-19) pandemic progression is a worldwide priority. CoronaVac is an inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine approved for emergency use with robust efficacy and immunogenicity data reported in trials in China, Brazil, Indonesia, Turkey, and Chile. Methods: This study is a randomized, multicenter, and controlled phase 3 trial in healthy Chilean adults aged ≥18 years. Volunteers received two doses of CoronaVac separated by 2 (0–14 schedule) or 4 weeks (0–28 schedule); 2302 volunteers were enrolled, 440 were part of the immunogenicity arm, and blood samples were obtained at different times. Samples from a single center are reported. Humoral immune responses were evaluated by measuring the neutralizing capacities of circulating antibodies. Cellular immune responses were assessed by ELISPOT and flow cytometry. Correlation matrixes were performed to evaluate correlations in the data measured. Results: Both schedules exhibited robust neutralizing capacities with the response induced by the 0–28 schedule being better. No differences were found in the concentration of antibodies against the virus and different variants of concern (VOCs) between schedules. Stimulation of peripheral blood mononuclear cells (PBMCs) with Mega pools of Peptides (MPs) induced the secretion of interferon (IFN)-γ and the expression of activation induced markers in CD4+ T cells for both schedules. Correlation matrixes showed strong correlations between neutralizing antibodies and IFN-γ secretion. Conclusions: Immunization with CoronaVac in Chilean adults promotes robust cellular and humoral immune responses. The 0–28 schedule induced a stronger humoral immune response than the 0–14 schedule. Funding: Ministry of Health, Government of Chile, Confederation of Production and Commerce & Millennium Institute on Immunology and Immunotherapy, Chile. Clinical trial number: NCT04651790 Editor's evaluation This manuscript investigates the humoral neutralizing antibody and cellular immune responses of volunteers in a randomized clinical trial for the CoronaVac SARS-CoV-2 vaccine. The findings are useful and provide context for the efficaciousness of the 0-14 day and 0-28 day dosing schedules of CoronaVac. The results show that these two dosing schedules are similar across most metrics. https://doi.org/10.7554/eLife.81477.sa0 Decision letter Reviews on Sciety eLife's review process Introduction The current coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Kim et al., 2020; Zhu et al., 2020), a virus described for the first time at Wuhan, China, in late December 2019. SARS-CoV-2 is already responsible for more than 530 million cases of infection and over 6 million deaths during the past 2 years (Dong et al., 2020). Worldwide efforts to develop effective vaccines against this virus have led to 10 vaccine prototypes approved for emergency use by the WHO, and over 8 billion vaccines administered to humans to date (World Health Organization, 2022). Most approved SARS-CoV-2 vaccines rely on a single viral component, namely the spike (S) protein or its receptor-binding domain (RBD), which could negatively impact the neutralizing capacities of antibodies induced if circulating VOCs, such as the Delta and Omicron, mutate those sequences (World Health Organization, 2022). Whole virus-inactivated platforms have been widely used throughout history to prevent diseases against other viruses (Kyriakidis et al., 2021). In the case of SARS-CoV-2, these vaccines contain a broader diversity of antigens than just the S protein. Therefore, they might be more suited to protect against emerging circulating variants (Kyriakidis et al., 2021; Melo-González et al., 2021). CoronaVac is an inactivated SARS-CoV-2 vaccine approved by the WHO for emergency administration to humans and developed by Sinovac Life Sciences Co., Ltd. (Gao et al., 2020; Mallapaty, 2021). Phase 1/2 trials in China confirmed that this vaccine induces a robust immune response against SARS-CoV-2 (Wu et al., 2021; Zhang et al., 2021). These data led to the evaluation of this vaccine in phase 3 clinical trials in other countries, including Brazil, Indonesia, Turkey, and Chile. An efficacy of 83.5% was reported for CoronaVac in Turkey and an effectiveness of 87.5% in Chile to prevent hospitalization due to COVID-19 for healthy adults (Jara et al., 2021; Tanriover et al., 2021). In this article, we compare the immune response elicited in healthy Chilean adults immunized with two doses of CoronaVac separated by either 2 (0–14 schedule) or 4 weeks (0–28 schedule). Our results suggest that, although the neutralizing capacities of antibodies elicited by a 0–28 immunization schedule with CoronaVac in Chilean adults are more robust than those induced by a 0–14 schedule, overall, both immune responses are equivalent. Materials and methods Study design, randomization, masking, and volunteers Request a detailed protocol This clinical trial (clinicaltrials.gov NCT04651790) was conducted in Chile at eight different sites, six located in Santiago city (Metropolitan Region) and two in the V Region of Valparaiso. The study protocol adhered to the current Tripartite Guidelines for Good Clinical Practices, the Declaration of Helsinki, and local regulations and was approved by the Institutional Scientific Ethical Committee of Health Sciences of the Pontificia Universidad Católica de Chile (#200708006). The execution was approved by the Chilean Public Health Institute (#24204/20). Recruited volunteers were adults aged ≥18 years, and informed consent was obtained upon enrollment. Volunteers received two doses of CoronaVac at day 0 and 2 (0–14) or 4 (0–28) weeks after the first immunization. Volunteers did not receive any payment for their participation. Study nurses oversaw the immunization and did not participate in any other study procedure. Inclusion and exclusion criteria were applied as previously reported (Bueno et al., 2022). Briefly, inclusion criteria considered being aged 18 and over, being able to understand and sign the informed consent form, and compliances with all study procedures and visits. Exclusion criteria considered mainly history of confirmed symptomatic SARS-CoV-2 infection, pregnancy, allergy to vaccine components, and immunocompromised conditions. Well-controlled medical conditions were allowed. Randomization was performed with a sealed enveloped system integrated with an electronic Case Report Forms (eCRF) in the OpenClinica platform; 2302 volunteers were enrolled in this phase 3 clinical trial by April 9, 2021. Four-hundred and forty volunteers were part of the immunogenicity arm; 199 of these were part of the 0–14 schedule; and 241 were part of the 0–28 schedule (Figure 1—figure supplement 1). The mean age of the recruited volunteers was 40.4±11.8 for the 0–14 schedule and 39.2±10.3 for the 0–28 schedule. A total of 52.9% volunteers were female and 47.1% were male. CoronaVac consists of 3 µg or 600SU of β-propiolactone-inactivated SARS-CoV-2 (strain CZ02) with aluminum hydroxide as an adjuvant in 0.5 mL (Gao et al., 2020). Sodium chloride, monosodium hydrogen phosphate, and disodium hydrogen phosphate are excipients, and water for injection is included as solvent. A study nurse administered ready-to-use syringes with 0.5 mL of CoronaVac intramuscularly in the deltoid area. Sera and PBMCs were isolated from blood obtained before administration of the first and the second dose and 2 and 4 weeks after the second dose for both immunization schedules. Procedures Request a detailed protocol For the isolation of sera, 20 mL of blood were collected in anticoagulant tubes and distributed in two tubes of 10 mL per volunteer (BD Vacutainer Clot Activator tubes #367896). Blood was allowed to clot for at least 1 hr at room temperature (RT). Samples were then centrifuged in a refrigerated centrifuge with a horizontal rotor at 1300× g for 10 min at 22°C. Serum was collected and stored at –80°C until use. Hemolyzed samples were rejected. For the isolation of PBMCs, blood was collected in three heparinized tubes (BD Vacutainer #367874, 10 mL) and stored at RT until processing. Samples were diluted with PBS (1:1) and centrifuged for 10 min at 1200× g (RT) in SepMate tubes (StemCell Technologies) with density-gradient medium (Lymphoprep). The plasma was then discarded and PBMCs were isolated by pouring them into a clean tube. Isolated PBMCs were washed twice with sterile PBS, counted, and cryopreserved in FBS (Industrial Biologicals) and 10% DMSO (Chem Cruz). All PBMC samples were stored in liquid nitrogen until use. To assess the presence of anti-SARS-CoV-2 antibodies, blood samples obtained before the first dose (preimmune), before the second dose, 2 and 4 weeks after the second dose were analyzed. The quantitative measurement of human IgG antibodies against the RBD domain of the S1 protein (S1-RBD) was determined using the RayBio COVID-19 (SARS-CoV-2) Human Antibody Detection Kit (Indirect ELISA method) (Cat #IEQ-CoVS1RBD-IgG) and through meso-scale discovery (MSD) immunoassays were performed as described previously (Johnson et al., 2020). Briefly, these kits consist of 96-well plates coated with the S1-RBD protein segment SARS-CoV-2. Sera samples were serially diluted starting at a 200-fold dilution until a 6400-fold dilution. After 1 hr of incubation at RT, the plates were washed, and a biotinylated anti-human IgG antibody provided in the kit was added and incubated for 30 min at RT. Plates were washed, and then a horseradish peroxidase (HRP)-conjugated streptavidin was added and incubated for 30 min at RT. Plates were rewashed, and the TMB substrate solution supplied in the kit was added. Finally, a stop solution provided in the kit was added, and absorbance was measured at 450 nm in an ELISA plate reader (Biotek, 1506021). As controls, dilutions of the First WHO International Standard for anti-SARS-CoV-2 immunoglobulin (human – NIBSC code: 20/13), and positive control provided by each kit were included. Additional controls included samples of volunteers seropositive or seronegative at recruitment or inoculated with placebo. To calculate the end titers, the cut-off value for each test was determined as ≥2.1 times the average OD450nm value of a panel of 8 serum samples from volunteers who received placebo and 12 seronegative serum samples. These serum samples were obtained before vaccination from seronegative volunteers. Seropositivity was determined as the highest dilution that reached >2.1 times the OD450nm cut-off value. Seroconversion was defined as an increase of at least four times the titer at baseline. For the surrogate virus neutralization test (sVNT), the SARS-CoV-2 Neutralizing Antibodies Test (BSNAT) Kit from BioHermes (COV-S41) was used to detect neutralizing antibodies in serum against SARS-CoV-2. This kit is a blocking assay which simulates the neutralization process, based on the ELISA platform. The main components of the kit are an ELISA plate pre-coated with the human ACE2 (hACE2) protein and the SARS-CoV-2 S1-RBD fragment conjugated with horseradish peroxidase (HRP-RBD). Briefly, the first step consisted in preparing serial dilutions from serum samples with the sample dilutor provided in the kit. Then, samples and controls were incubated with the HRP-RBD for 10 min at 37°C to allow the binding of neutralizing antibodies to RBD. Sera and controls previously incubated with HRP-RBD were added to ELISA plates (pre-coated with hACE2) and incubated for 20 min at 37°C. After incubation, samples were discarded, and all the wells were washed five times. Finally, TMB solution was added and quenched after 15 min of incubation at RT. Plates were read at 450 nm in a microplate reader (EPOCH, Biotek 1506021). The neutralizing antibody titer was determined as the last fold dilution with a cut-off value over 30% of inhibition. The inhibition rate was calculated based on the negative control absorbances (negative control – sample/negative control*100), and 10% was considered as the cut-off value. A similar methodology was performed to evaluate the neutralizing antibodies in sera against different VOCs, as reported previously (Melo-González et al., 2021). For the conventional virus neutralization test (cVNT), Vero E6 cells were infected with a SARS-CoV-2 strain obtained by viral isolation in tissue cultures (33782CL-SARS-CoV-2 strain). Neutralization assays were carried out by the reduction of cytopathic effect (CPE) in Vero E6 cells (ATCC CRL-1586, confirmed to be Mycoplasma free). The titer of neutralizing antibodies was defined as the highest serum dilution that neutralized virus infection, at which the CPE was absent compared with the virus control wells (cells with CPE). Vero E6 cells (4×104 cells/well) were seeded in 96-well plates. For neutralization assays, 100 µL of 33782CL-SARS-CoV-2 (at a dose of 100 TCID50) were incubated with serial dilutions of heat-inactivated sera samples (dilutions of 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, and 1:512) from volunteers for 1 hr at 37°C. Then, the mix was added to the 96-well plates with the Vero E6 cells. CPE on Vero E6 cells was analyzed 7 days after infection. For each test, a serum sample from uninfected patients (negative control) and a neutralizing COVID-19 patient serum sample (positive control) were used. For the pseudotyped virus neutralization test (pVNT), anti-SARS-CoV-2 neutralizing antibodies were measured using an HIV-1 backbone expressing firefly luciferase as a reporter gene and pseudotyped with the SARS-CoV-2 spike glycoprotein (HIV-1-SΔ19) as previously described (Beltrán-Pavez et al., 2021). Briefly, serum samples were initially diluted 1:4 in DMEM, serially diluted 1:3 up to 1:8748, and then mixed with approximately 4.5 ng of p24 equivalents of HIV-1-SΔ19 in white 96-well plate. Plates were incubated for 1 hr at 37°C, and then 100 mL of DMEM containing 1×104 HEK-ACE2 (transformed from CRL-3216 ATCC, confirmed to be Mycoplasma free) cells was added to each well. Firefly luciferase activity was measured 48 hr later, using the Luciferase Assay System (Promega) in a Glomax-96 microplate luminometer (Promega). Estimation of the ID50 was obtained using a four-parameter nonlinear regression curve fit measured as the percent of neutralization determined by the difference in average relative light units between test samples and pseudotyped virus controls as previously described (Beltrán-Pavez et al., 2021). Data analyses and statistical analyses were carried out using GraphPad Prism v.9. To assess the cellular immune response, ELISPOT and flow cytometry assays were performed using PBMCs from volunteers at the different times indicated above. Upon thawing, cells were resuspended in fresh media in a 1:10 dilution to remove DMSO remnants from the freezing media. Then, cells were centrifuged, resuspended in fresh media, and counted in an automated cell counter (Logos Biosystems #L40001). Cells were adjusted to 6×106 cells/mL and kept at 37°C, 5% CO2 for 15 min until use in the corresponding assay. ELISPOT plates containing a PVDF membrane were activated with 15 µL of 70% ethanol (Merck), washed three times with sterile ×1 PBS, and then coated with human IFN-γ and IL-4 capture antibodies (1:250 and 1:125, respectively, CTL). After 3 hr of activation at RT, plates were washed two times with PBS and two times with PBS-Tween 20 0.05%. The stimulus included in these assays considers the use of Mega Pools (MPs) of peptides derived from SARS-CoV-2 proteins, previously described (Grifoni et al., 2020). Two MPs composed of peptides from the S protein (MP-S) and the remaining proteins of the viral particle (MP-R) were used, as previously described (Grifoni et al., 2020). These peptides were determined in silico to stimulate CD4+ T cells optimally. Also, two MPs composed of peptides from the proteome of SARS-CoV-2 (CD8-A and CD8-B) were used, as previously described (Grifoni et al., 2020). These peptides were determined in silico to optimally stimulate CD8+ T cell. As positive controls, an independent stimulation performed with 5 mg/mL of Concanavalin A (ConA) (Sigma Life Science #C5275-5MG), and with an MP of peptides derived from cytomegalovirus proteins (MP-CMV) for the stimulation of both CD4+ and CD8+ T cells (Grifoni et al., 2020). As a vehicle control, DMSO 1% (Merck #317275) was included. A total of 3×105 cells in 50 µL of media were added to each well containing 50 µL of media with the corresponding stimulus. The final concentration of each stimulus per well was 1 µg/mL (except for ConA and DMSO). Positive controls for ELISPOT assays considered 5×104 cells/well instead of 3×105 cells/well. For ELISPOT assays, cells were incubated for 48 hr at 37°C, 5% CO2. After incubation, plates were washed one time with PBS, and three times with PBS-Tween 20. Then, anti-human IFN-γ (FITC) and anti-human IL-4 (Biotin) antibodies (1:1000 and 1:1000, respectively) were added, and plates were incubated for 2 hr, RT. Plates were washed three more times with PBS-Tween 20 and then FITC-HRP and Streptavidin-AP (1:1000) were added and plates were incubated for 1 hr, RT. After incubation, plates were washed three more times with PBS-Tween 20. Then, plates were treated with the blue (15 min), and red (15 min) developer solution individually following the recommendations of the manufacturer. Plates were washed with tap water after each developer solution and allowed to dry for 24 hr prior to reading. To evaluate the number of T cells secreting IFN-γ, IL-4, or both, ELISPOT assays were performed with ImmunoSpot technology (ImmnunoSpot #hIFNgIL4-1M-10). Spot forming cells (SFCs) were counted on an ImmunoSpot S6 Micro Analyzer. To characterize the expression of activation-induced markers (AIM) by T cells, flow cytometry assays were performed. 3×105 cells/well were stimulated as described for the ELISPOT assays, and after 24 hr of incubation with the stimulus, samples were stained. Staining was performed by incubation for 45 min at 4°C using the following reagents: a mix of BD Horizon Fixable Viability Stain 510 (BD Biosciences – CAT 564406–1 µL per 1×106 cells); anti-CD14 V500 (BD Biosciences – clone M5E2); anti-CD19 V500 (BD Biosciences – clone HIB19); anti-CD3 AF-700 (Biolegend – clone OKT3); anti-CD69 PE (BD Biosciences – clone FN50); anti-CD8a BV-650 (BD Biosciences – clone RPA-T8); anti-CD4 BV-605 (BD Biosciences – clone RPA-T4); anti-CD137 (BioLegend, Clone 4-1BB); and anti-OX40 (BioLegend, Clone BER-ACT35). Cells were washed twice with 200 µL of PEB buffer, fixed, and then handed to the Flow Cytometry core facility, for their acquisition in an LSRFortessa X-20 flow cytometer. Statistical analyses Request a detailed protocol Sample size determination was already reported for this trial (Bueno et al., 2022). Statistical significance was set at α=0.05 in all cases. Statistical analyses and symbols used for each analysis are described briefly in each figure legend. All statistical analyses were performed in GraphPad Prism v.9.0.1 or RStudio. To evaluate statistical differences of anti-S antibody titers and neutralizing antibody titers either by cVNT, sVNT, or pVNT induced by either immunization schedule, a two-tailed one-way ANOVA for repeated measures was performed over the Log2 of antibody titers, followed by Bonferroni’s multiple comparisons test in order to compare both schedules. Differences in seroconversion rates of neutralizing antibody titers were determined by a two-tailed Fisher’s exact test. Volunteers that were detected to be seropositive at entry for anti-N and/or anti-S antibodies were excluded from the analyses. For the ELISPOT data, comparison between schedules was performed by a two-tailed one-way ANOVA for repeated measures which was performed over the Log2 of antibody titers, followed by Bonferroni’s multiple comparisons test over the Log10 of the fold change. Differences among schedules for flow cytometry data were assessed by a two-tailed one-way ANOVA for repeated measures which was performed over the Log2 of antibody titers, followed by Bonferroni’s multiple comparisons test of the percentage data. Pearson correlation matrixes were generated for each immunization schedules considering both humoral and cellular immune response data. Humoral response data were transformed to the base 10 logarithm before analysis. Individual Pearson correlations were selected based on their immunological relevance, n, r, and p values. These values, as well as the 95% confidence bands of the correlation, are shown in each graph. Other comparisons assessed were analyzed via two-tailed unpaired t tests, two-tailed non-parametric unpaired t tests (Mann-Whitney tests), two-tailed non-parametric paired t tests (Wilcoxon tests), ANOVA followed by Tukey tests, or two-tailed Fisher’s exact test, as indicated in the figure legends. Results A 0–28 day immunization schedule with CoronaVac promotes higher seropositivity rates and GMT values of neutralizing antibodies than a 0–14 schedule To evaluate the humoral immune response elicited after vaccination with two doses of CoronaVac, separated by 2 or 4 weeks (0–14 and 0–28 days schedules, respectively), the neutralizing capacities of circulating antibodies were evaluated. This was performed independently through an sVNT (Figure 1A and C) and cVNT for the Ancestral strain (Figure 1B and D), as well as a pseudotyped virus neutralization test (pVNT) (Figure 1—figure supplement 2). For both immunization schedules, samples from 130 volunteers were tested for sVNT, 372 volunteers for cVNT with the Ancestral strain, and 94 for pVNT (Table 1). These techniques show a robust increase in arbitrary WHO international units (IU), geometric mean titer (GMT) values, and seropositivity rates 2 and 4 weeks after the second dose for both immunization schedules. Remarkably, as seen in IU for the sVNT and GMT values for cVNT and pVNT, the 0–28 schedule showed increased neutralizing capacities 2 and 4 weeks after the second dose (Figure 1A, B and Figure 1—figure supplement 2A). No differences in seropositivity rates between both schedules were detected for any of the assays evaluated (Figure 1C, D and Figure 1—figure supplement 2B). We also evaluated differences in the neutralizing capacities of circulating antibodies between the two age groups indicated for all four techniques (Figure 1—figure supplement 3). Both age groups had significantly increased GMT values at all times compared to preimmune samples, irrespective of the immunization schedule and the technique evaluated (Table 2). However, we observed significantly higher titers of circulating neutralizing antibodies in the 18–59 years age group compared to the >60 years age group, 2 and 4 weeks after the second dose, irrespective of the vaccination schedule, as determined by cVNT and pVNT (Figure 1—figure supplement 3 and Table 1). These results suggest that CoronaVac induces the production of circulating antibodies with varying neutralizing capacities after immunization with either a 0–14 or a 0–28 schedule. Remarkably, the 0–28 schedule promotes higher seroconversion rates and GMT or IU values of these neutralizing antibodies than the 0–14 schedule, as determined by sVNT and pVNT. Figure 1 with 3 supplements see all Download asset Open asset Circulating neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) measured by surrogate virus neutralization test (sVNT) and conventional virus neutralization test (cVNT) for the Ancestral strain in immunized volunteers. Neutralizing antibody titers were evaluated with an sVNT, which quantifies the interaction between S1-RBD and human ACE2 (hACE2) pre-coated on ELISA plates (A,C) and with a cVNT, which quantifies the cytopathic effect (CPE) induced in Vero cells as plaques formation (B, D). n=372 volunteers for cVNT (Ancestral) and n=130 volunteers for sVNT (for both schedules). Data is represented as the reciprocal antibody titer of neutralizing antibody versus the different times evaluated. Numbers above the bars show either the arbitrary international units (IU) (A) or the geometric mean titer (GMT) (B), and the error bars indicate the 95% CI. Seropositivity rates are also displayed (C, D). Data from IU and GMT values were analyzed by a two-tailed unpaired t-test of the base 2 logarithms of data to compare immunization schedules. Data from seropositivity rates were analyzed by a two-tailed Fisher’s exact test. Numbers above each bracket represent calculated p values comparing both immunization schedules. Statistical significance was set at p<0.05 and highlighted numbers indicate statistical significance. Figure 1—source data 1 Data used to generate Figure 1, Figure 1—figure supplement 3, and Figure 5. https://cdn.elifesciences.org/articles/81477/elife-81477-fig1-data1-v2.xlsx Download elife-81477-fig1-data1-v2.xlsx Table 1 Seropositivity rates and geometric mean titer (GMT) values measured for circulating neutralizing antibodies induced by CoronaVac in both immunization schedules and dissected by age group. Antibodies evaluatedScheduleAge groupIndicators2 Weeks after second dose*p Value4 Weeks after second dose†p ValuesVNT0–14 daysTotal vaccineSeropositivity n/N41/530.01847/570.1100%77.482.5IU/mL50.951.295% CI37.4–59.138.5–68.018–59 yearsSeropositivity n/N30/3229/32%93.890.6IU/mL76.068.895% CI50.2–115.146.0–102.9≥60 yearsSeropositivity n/N11/2118/25%52.372.0IU/mL27.635.195% CI20.0–38.124.2–50.80–28 daysTotal vaccineSeropositivity n/N66/730.03566/730.1100%90.490.4IU/mL178.2131.795% CI133.5–238.1100.7–172.318–59 yearsSeropositivity n/N33/3333/33%100100IU/mL255.9178.295% CI175.3–373.7123.6–256.9≥60 yearsSeropositivity n/N33/4033/40%82.582.5IU/mL132.0102.695% CI87.0–200.570.0–150.3cVNT (Ancestral)0–14 daysTotal vaccineSeropositivity n/N147/150<0.0001156/160<0.0001%98.097.5GMT46.933.895% CI38.6–57.028.4–40.218–59 yearsSeropositivity n/N121/121128/128%100100GMT59.242.695% CI48.8–71.835.7–50.9≥60 yearsSeropositivity n/N26/2928/32%89.787.5GMT17.313.195% CI10.7–28.08.9–19.10–28 daysTotal vaccineSeropositivity n/N208/210<0.0001209/212<0.0001%99.098.6GMT90.261.195% CI76.8–105.852.3–71.418–59 yearsSeropositivity n/N124/125123/124%99.299.2GMT121.982.495% CI102.5–144.969.6–97.7≥60 yearsSeropositivity n/N84/8586/88%98.897.7GMT57.940.195% CI43.7–76.630.7–52.4pVNT0–14 daysTotal vaccineSeropositivity n/N73/77<0.000173/770.0027%97.397.3GMT52.740.195% CI36.6–76.428.9–55.918–59 yearsSeropositivity n/N48/4948/49%97.997.9GMT83.359.095% CI53.6–129.539.3–88.7≥60 yearsSeropositivity n/N25/2825/28%89.289.2GMT23.120.095% CI13.2–40.712.3–32.50–28 daysTotal vaccineSeropositivity n/N16/170.000816/170.0029%94.194.1GMT146.7104.995% CI60.0–359.041.9–262.618–59 yearsSeropositivity n/N8/88/8%100100GMT505.9328.795% CI306.1–836.0159.6–676.8≥60 yearsSeropositivity n/N8/98/9%88.888.8GMT48.838.095% CI13.3–178.69.56–152.0 Red values indicate statistically significant results (p<0.05). * p Values are for comparison of IU/mL or GMT levels between 18 and 59 years and >60 years age groups 2 weeks after the second dose. † p Values are for comparison of IU/mL or GMT levels between 18 and 59 years and >60 years age groups 4 weeks after the second dose. Table 2 p Values estimated for neutralization assays evaluated for both immunization schedules. FigureParameter evaluatedSchedule;preimmune valuePreimmune compared to2 Weeks after second dose4 Weeks after second doseFigure 1AsVNT(IU/mL;p value)0–14 days;16.450.9;<0.000151.2;<0.00010–28 days;17.7178.2;<0.0001131.7;<0.0001Figure 1BcVNT (Ancestral)(GMT;p value)0–14 days;2.0446.9;<0.000133.8;<0.00010–28 days;2.0890.2;<0.000161.1;<0.0001Figure 1—figure supplement 2pVNT(GMT;p value)0–14 days;2.0552.66;<0.000140.14;<0.00010–28 days;2.0146.71;<0.0001104.93;<0.0001 *p Values were determined by performing one-way ANOVAs for repeated measures over Log10 of data, followed by post hoc Bonferroni's multiple comparisons test. Red values indicate statistically significant results (p<0.05). Both immunization schedule with CoronaVac exhibits similar levels of anti-S1 and anti-RBD-specific antibodies To evaluate the humoral immune response elicited after vaccination with two doses of CoronaVac, separated by 2 or 4 weeks (0–14 and 0–28 day schedules, respectively), antibody titers against the Ancestral S1 and the RBD of SARS-CoV-2 were evaluated before administration of the first and second dose, and 2 and 4 weeks after the second dose (Figure 2). Samples from 162 volunteers were assessed independently for the S1-RBD through ELISA assays (Figure 2A) and 44 through MSD immunoassays (Figure 2B). Circulating antibodies against the S1-RBD were robustly increased for both immunization schedules at all times evaluated after administration of the first dose (preimmune), as determined by geometric mean units (GMUs) values of the arbitrary WHO international standard. No differences were found at all times evaluated for anti-S1-RBD-specific antibodies between both schedules (Figure 2A). Accordingly, no differences could be found between schedules for the MSD analyses performed, either for the S protein (left) or the RBD (right) of the Ancestral strain of SARS-CoV-2 (Figure 2B). These results show that CoronaVac induces a statistically significant increase in anti-S1 and anti-RBD antibodies after immunization with either a 0–14 or a 0–28 sched

Author Response: Differences in the Immune Response Elicited by Two Immunization Schedules with an Inactivated SARS-CoV-2 Vaccine in a Randomized Phase 3 Clinical Trial