ORIGINAL RESEARCH published: 31 January 2022 doi: 10.3389/fimmu.2021.766278 Evaluation of the Immune Response Induced by CoronaVac 28-Day Schedule Vaccination in a Healthy Population Group Alejandro Escobar 1†, Felipe E. Reyes-López 2,3,4†, Mónica L. Acevedo 5, Luis Alonso-Palomares 5, Fernando Valiente-Echeverrı́a 5, Ricardo Soto-Rifo 5, Edited by: Hugo Portillo 1, Jimena Gatica 1, Ivan Flores 1, Estefanı́a Nova-Lamperti 6, Arun Kumar, Coalition for Epidemic Preparedness Carlos Barrera-Avalos 2, Marı́a Rosa Bono 7, Leonardo Vargas 7, Valeska Simon 7, Innovations (CEPI), Norway Elias Leiva-Salcedo 2, Cecilia Vial 8, Juan Hormazabal 8, Lina Jimena Cortes 8, Daniel Valdés 2, Ana M. Sandino 2, Mónica Imarai 2* and Claudio Acuña-Castillo 2* Reviewed by: Rupsa Basu, 1Laboratorio Biologı´a Celular y Molecular, Instituto de Investigación en Ciencias Odontológicas, Facultad de Odontologı´a, Helaina Inc., United States Universidad de Chile, Santiago, Chile, 2 Centro de Biotecnologı´a Acuı´cola, Facultad de Quı´mica y Biologı´a, Universidad de Abhishek Mishra, Santiago de Chile, Santiago, Chile, 3 Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Lala Lajpat Rai Memorial Medical Barcelona, Bellaterra, Spain, 4 Facultad de Medicina Veterinaria y Agronomía, Universidad de Las Américas, Providencia, College, India Chile, 5 Laboratory of Molecular and Cellular Virology, Virology Program, Institute of Biomedical Sciences, Faculty of *Correspondence: Medicine, Universidad de Chile, Santiago, Chile, 6 Molecular and Translational Immunology Laboratory, Department of Clinical Mónica Imarai Biochemistry and Immunology, Faculty of Pharmacy, University of Concepcion, Concepcion, Chile, 7 Departamento de monica.imarai@usach.cl Biologı´a, Facultad de Ciencias, Universidad de Chile, Santiago, Chile, 8 Programa Hantavirus y Zoonosis, Instituto de Claudio Acuña-Castillo Ciencias e Innovación en Medicina, Facultad de Medicina, Clı´nica Alemana Universidad del Desarrollo, Santiago, Chile claudio.acuna@usach.cl † These authors have contributed CoronaVac vaccine from Sinovac Life Science is currently being used in several countries. equally to this work In Chile, the effectiveness of preventing hospitalization is higher than 80% with a Specialty section: vaccination schedule. However, to date, there are no data about immune response This article was submitted to induction or specific memory. For this reason, we recruited 15 volunteers without previous Vaccines and Molecular Therapeutics, a section of the journal suspected/diagnosed COVID-19 and with negative PCR over time to evaluate the Frontiers in Immunology immune response to CoronaVac 28 and 90 days after the second immunization (dpi). Received: 28 August 2021 The CoronaVac administration induces total and neutralizing anti-spike antibodies in all Accepted: 14 December 2021 vaccinated volunteers at 28 and 90 dpi. Furthermore, using ELISpot analysis to assay Published: 31 January 2022 cellular immune responses against SARS-CoV-2 spike protein, we found an increase in Citation: Escobar A, Reyes-López FE, IFN-gamma- and Granzyme B-producing cells in vaccinated volunteers at 28 and 90 dpi. Acevedo ML, Alonso-Palomares L, Together, our results indicate that CoronaVac induces a robust humoral immune Valiente-Echeverrı´a F, Soto-Rifo R, Portillo H, Gatica J, Flores I, response and cellular immune memory of at least 90 dpi. Nova-Lamperti E, Barrera-Avalos C, Keywords: CoronaVac, SARS-CoV-2, herd immunity, neutralizing antibodies, COVID-19, vaccine, immunological memory Bono MR, Vargas L, Simon V, Leiva-Salcedo E, Vial C, Hormazabal J, Cortes LJ, Valdés D, Sandino AM, Imarai M and Acuña-Castillo C (2022) Evaluation of the Immune INTRODUCTION Response Induced by CoronaVac 28-Day Schedule Vaccination The pandemic of coronavirus disease 2019 (COVID-19) has led to an unprecedent rapid vaccine in a Healthy Population Group. development and production aimed to prevent the spread of the SARS-CoV-2 virus. The clinical Front. Immunol. 12:766278. severity of the disease has led to the urgent application and approval of vaccines with phase 3 studies doi: 10.3389/fimmu.2021.766278 under development, with proven safety results, but with little data regarding immune response, Frontiers in Immunology | www.frontiersin.org 1 January 2022 | Volume 12 | Article 766278 Escobar et al. CoronaVac 28-Day Schedule Vaccination efficacy, and mechanism of action to prevent the disease. In this studies, about 30 volunteers were recruited. Inclusion criteria scenario, the immune mechanisms triggered by SARS-CoV-2 included the absence of suspected/diagnosed case of COVID-19 vaccines in healthy populations are not well-known. Studies are and testing negative for COVID-19 for at least six previous PCR limited, especially from laboratories not directly related to the analyses in monthly screening tests. In addition, immunological manufacture of vaccines or research teams leading the diseases or any associated chronic disease with no treatment were evaluations of phase 2 or 3 clinical trials (1). considered exclusion criteria. Blood samples were obtained before Currently, in Chile, two types of vaccines have been massively the vaccination process. The Scientific Ethics Committee approved applied, the BNT162b2 vaccine by Pfizer-BioNTech vaccine the study and informed consent, Universidad de Santiago de Chile generated from engineered messenger RNA (mRNA) encoding (172/2021). In addition, written informed consent was obtained the spike protein (S) of SARS-CoV-2 (2), and the CoronaVac from each enrolled participant. Paired samples were analyzed vaccine from Sinovac Life Sciences that contains the inactivated using Friedman and Quade test with multiple comparison post- SARS-CoV-2 virus (3). A recent report in the Chilean population test. Unpaired data analysis was performed using Mann–Whitney vaccinated with two doses of CoronaVac separated by 14 days test. All statistical analyses were accomplished with GraphPad showed a seroconversion rate for anti-S1-RBD (receptor binding Prism version 8.01 (GraphPad Software Inc, La Jolla, CA). The domain) IgG of 18.1%, 100%, and 87.5% at 14, 28, and 42 days results were presented as median. p < 0.05 was considered post-immunization (dpi), respectively. Moreover, a 95.7% statistically significant. seroconversion rate was reported for neutralizing antibodies in the 18- to 59-year-old group after 28 and 42 dpi, and the Sample Processing induction of a T-cell response characterized by the secretion of Blood was collected in BD Vacutainer tubes (REF367281, Becton, interferon (IFN)-gamma (IFN-g) upon stimulation with SARS- Dickinson and Company, USA) by health-trained personnel. CoV-2 Mega Pools of peptides was also observed (4). The samples obtained (15 ml at each time point) were coded, The current COVID-19 mass vaccination schedule consists of kept cold, and processed to obtain plasma and peripheral blood two doses of the vaccines separated by 4 weeks (28 days apart mononuclear cells (PBMCs). One milliliter of blood was schedule vaccination) (5). Recently, a prospective national cohort aliquoted to obtain blood plasma by centrifugation. Then, 300 study reported that using this vaccination schedule, CoronaVac µl was transferred to cryopreservation tubes and frozen at −80°C is effective for preventing hospitalization (87.5%), Intensive Care until use. White cells were isolated by Ficoll-Hypaque gradient Unit (ICU) admission (90.3%), and Covid-19–related death centrifugation (Gibco BRL lymphoprep). (86.3%) (6). Many questions have arisen about the need of a third booster dose, especially given the absence of detailed and Production and Purification Spike Protein conclusive evidence of the immune responses induced by from SARS-CoV-2 CoronaVac using the 28-day-apart vaccination. Therefore, in The sequence coding for the spike protein modified by Amanat this study, we evaluated the production of neutralizing et al. (GenBank: MN908947.3) was commercially synthesized antibodies, the activation of the cellular response, and the and cloned into the pcDNA3.1 plasmid (GenScript, Piscataway, generation of cellular memory induced after CoronaVac 28- NJ, USA). The transfection was performed using the ExpiCHO day schedule vaccination in a healthy population group Expression System. Briefly, the ExpiCHO-S cells were cultured in independently of the production laboratory. Together, our a vented flask with ExpiCHO Expression medium (Thermo results indicate that CoronaVac induces a robust humoral Fisher, Cat. A2910001) in a humidified 8% CO2 incubator at immune response and cellular immunity of at least 90 dpi, 37°C at 400g and passaged every 3–4 days at 200,000 cells/ml, which can explain prevention of COVID19 with severe until transfection day. The day before transfection, the cells were symptoms (6, 7). split at 3 × 106 cells/ml. After 24 h, the cells were diluted at 6 × 106 cells/ml with a fresh medium. The ExpiCHO transfections were performed using the ExpiCHO Expression System Kit MATERIALS AND METHODS (Thermo Fisher, Cat. A29133), according to the manufacturer’s protocol, and cultures were then temperature shifted to 32°C and Study Population, Study Design, and cultured for 5 to 7 days. Then, cell suspension was centrifuged at Statistical Analysis 3,220g for 20 min at 4°C to pellet cells. The supernatant was Twenty-one adult volunteers (mean age, 36 years old [range 27 to collected and centrifuged at 5,000g for 30 min, the pH was 61]) who were scheduled to receive the CoronaVac vaccine were adjusted to 8.0, and inhibitor proteases were added. The recruited from professionals from the area of clinical laboratories supernatant was dialyzed in native dialysis buffer (50 mM for COVID detection at the Universidad de Santiago de Chile Tris–HCl, pH 8.0, 250 mM NaCl) overnight at 4°C. The during March 2021. The sample size was calculated according to purification was performed by exclusion chromatography in the associated hypothesis using the G test. Since a 60% protection Ni-NTA (Gold Bio, Cat H-350-100). Briefly, columns were has been described for this vaccine, a 60% induction of memory at equilibrated using native binding buffer (50 mM Tris–HCl, pH 90 days post-vaccination was expected. This analysis showed that 8.0, 250 mM NaCl, 10 mM Imidazole). The supernatant was the minimum sample size was 15 volunteers. Because usually, loaded onto the equilibrated columns and incubated for 2 h at volunteers declined their participation without completing the 4°C in an orbital shaker. Following incubation, the supernatant Frontiers in Immunology | www.frontiersin.org 2 January 2022 | Volume 12 | Article 766278 Escobar et al. CoronaVac 28-Day Schedule Vaccination was collected, and the columns were washed twice with native and plasmids pNL4.3-DEnv-Firefly and pCMV14–3X-Flag– wash buffer (50 mM Tris–HCl, pH 8.0, 250 mM NaCl, 20 mM SARS-CoV-2 SD19C in a 1:1 ratio as we recently described (8). Imidazole). Next, the protein was eluted in the elution buffer (50 Cell culture media was replaced and collected at 16 h and 48 h mM Tris–HCl, pH 8.0, 250 mM NaCl, 250 mM Imidazole) and post-transfection, respectively, and centrifugated to 850g for concentrated in an Amicon Ultra-15 centrifugal filter unit 5 min at room temperature. Viral particles were diluted with (50,000 NMVL) at 3,220g at 4°C for 15 min. The buffer was 50% in fetal bovine serum (Sigma-Aldrich) and stored at −80°C. discarded, and the protein was washed twice using storage buffer Viral stock was quantified with the HIV-1 Gag p24 Quantikine (20 mM Tris–HCl, pH 8.0, 150 mM NaCl). Proteins were ELISA kit (R&D Systems) and titrated by serial dilutions. analyzed by reducing SDS-PAGE, dyeing with Sypro Ruby Neutralization assays were performed as we previously (Thermo Fisher, Cat. S12001), and quantified using ImageJ reported (8). Briefly, inactivated serum samples were diluted in software. Finally, the Spike protein was aliquoted and stored at DMEM with 10% fetal bovine serum and incubated with 3 to 5 −80°C, until use. ng of p24 of HIV-1-based SARS-CoV-2 pseudotyped particles. After 1 h incubation at 37°C, 1 × 104 HEK-ACE2 cells were Cytokines added to each well. HEK293T cells incubated with the Analyses were performed in plasma samples obtained from the pseudotyped virus were used as a negative control. Cells were volunteers, before vaccination and 28 days after the second lysed 48 h later, and firefly luciferase activity was measured using immunization. The cytokines TNF-a, IL-12, IL-6, IL-10, IL-1b, the Luciferase Assay Reagent (Promega) in a Glomax 96 and IL-8 were quantified using the Cytometric Bead Array (CBA) Microplate luminometer (Promega). The percentage of System (Becton Dickinson, BD-USA). Data acquisition was neutralization for each dilution was calculated, and achieved by flow cytometry using a FacsCanto II flow neutralization titers, defined as 50% inhibitory dose (ID50), cytometer (BD-USA) and analyzed in the BD FCAP Array were estimated as previously described (8). Briefly, ID50 for software, where values were expressed in pg/ml for each cytokine. each sample was obtained using a four-parameter non-linear regression. Pass/fail quality control criteria for the assay were as Total Anti-Spike Antibodies follows (1): the average RLUs of pseudotype control wells are ≥10 Total antibody levels were determined using a previously established times the average RLUs of negative control (2); the % coefficient protocol (https://doi.org/10.1038/s41591-020-0913-5). Briefly, of variation (CV) of RLUs for triplicate wells control is ≤30% (3); ninety-six-well plates were coated at 4°C with 50 ng/well of the the % CV of duplicate is ≤30% for sample dilutions that yield at recombinant SARS-COV-2 S protein S1 (Biolegend 792906) in PBS. least 40% neutralization; and (4) positive control neutralization After overnight incubation, the coating solution was removed and curve crosses the 50% neutralization cutoff 0 to 1 times. All 100 ml/well of PBS, containing 0.1% Tween-20 (PBST) and 3% non- statistical analyses were performed using GraphPad Prism fat milk, were added to the plates. Plates were incubated for 1 h at version 8.0.1 software. room temperature. The serum samples were previously heated at 56°C for 1 h, and serial dilutions of serum were prepared in 1% non- fat milk prepared in PBST. One hundred microliters of each serial Enzyme-Linked Immunospot The specific activation of T lymphocytes was evaluated by IFN-g dilution was added, and the plates were incubated for 2 h at room and Granzyme B kit (R&D system). PBMCs were isolated by temperature. The plates were washed three times using 0.1% PBST using Ficoll-Hypaque gradient centrifugation (Lymphoprep and then 100 ml of a 1:10,000 dilution of donkey anti-human IgG– Gibco BRL) and leucocyte cell numbers were quantified using horseradish peroxidase (HRP) conjugated antibody (Clone Trypan Blue. Anti–IFN-g and Anti-Human Granzyme B Poly24109, Biolegend 410902) was added to each well for 1 h. antibody-coated wells (R&D Systems) were seeded with Plates were again washed with 0.1% PBST and then dried. Next, 300,000 leucocyte cells per well. Cells were stimulated in 100 ml of TMB (3,3′,5,5′-Tetramethylbenzidine BD OptEIA) duplicate with 10 µM SARS-CoV-2 spike structural protein solution was added to each well. This substrate was left on the (1.3 mg/ml). OKT3 and CD28 were used as positive control of plates for 10 min and then the reaction was stopped by the addition stimulation (3 and 5 mg/ml, respectively). Culture medium was of 50 ml per well of 1 M Phosphoric acid. The optical density at 490 the negative control. PBMCs were cultured for 48 h before nm was measured using an Emax (Molecular Devices, USA) plate enzymatic revelation of IFN-g and Human Granzyme B (R&D reader. Results are calculated as the area under the curve of four- Systems). Spots were counted by using an ELISPOT reader serial dilutions of patient plasma obtained by the ELISA technique. (A.EL.VIS ELISPOT Scanner) and results are expressed as the mean number of spot-forming units/3 × 106 peripheral blood IgM and IgG mononuclear cells after subtraction of the background value. Rapid tests (OnSite Covid-19 IgG/IgM Rapid Test-CTK Biotech, Inc, USA) were used according to manufacturer’s instructions to detect antibodies before vaccination, and at 28, 48, 78, 108, and 138 dpi. RESULTS Neutralizing Antibodies Characteristics of the Study Population Pseudotyped viral particles were produced by transient We evaluated the immune response to the CoronaVac vaccine transfection of HEK293T cells using polyethylenimine (PEI) applied in the Chilean population following an interval of Frontiers in Immunology | www.frontiersin.org 3 January 2022 | Volume 12 | Article 766278 Escobar et al. CoronaVac 28-Day Schedule Vaccination 28 days between the first and second dose schedule. We whom samples were not always collected, the increase in anti-S measured the capability of the vaccine to activate the humoral antibody levels showed the same kinetics (Figure 2B). and cellular responses in volunteers without suspected/ Additionally, we measured SARS-CoV-2 neutralizing diagnosed COVID-19 and with negative PCR over time antibodies (NAbs) using the HIV-1–SD19 pseudotype (8). We throughout the whole study. Table 1 shows the demographic found no volunteers with anti-spike neutralizing response before and baseline characteristics of the enrolled volunteers. We immunization (0 dpi = 4 ± 0.7, Figure 3A). In contrast, after 28 assessed the presence of anti-SARS-CoV-2 antibodies using and 90 dpi, we found anti-S neutralizing antibodies in all rapid detection tests before the first dose of CoronaVac, and volunteers in different levels (28 dpi = 388.1 ± 454.5, 90 28, 48, 78, 108, and 138 dpi. We found that all volunteers (21 dpi = 57.1 ± 67.6, Figures 3B, C). All 15 volunteers (complete subjects) showed anti-SARS-CoV-2 IgG at 28 dpi, and one (5%) follow-up) show an increase in the IC50 after 28 dpi (Figure 3D). showed specific IgM too. After 108 dpi, one volunteer showed no Furthermore, after 90 dpi, the neutralizing antibody levels IgG, and 138 dpi, we found 3 volunteers present no IgG. declined; however, some volunteers still showed this type of antibodies. Importantly, these results are consistent with those The Vaccine Triggers the Secretion of observed for the volunteers with a not complete follow- Interleukin 8 up (Figure 3E). The innate immune response is critical for vaccination as it activates the myeloid immune cells to release inflammatory mediators and initiate adaptive immunity the primary and CoronaVac Induces Immune Cellular post-boost immunization (9). To assess the pro- and anti- Response inflammatory plasma cytokine profile, we performed flow To explore the cellular immune responses to SARS-CoV-2 after cytometry and found that, 28 dpi, only IL-8 increased its level CoronaVac vaccination, we isolated PBMCs from the 15 from 14.7 ± 2.6 (on day 0) to 55.9 ± 63.8 pg/ml (on 28 dpi) volunteers with complete follow-up, and the PBMCs obtained (Figure 1). No significant changes were observed between 0 and were stimulated in vitro with the SARS-CoV-2 spike (S) protein. 28 dpi in the levels of tumor necrosis factor (TNF)-a (4.7 ± 9.5 to We observed a significant increase in the number of IFN-g spot- 4.7 ± 5.1 pg/ml), interleukin (IL)-12 (6.4 ± 20.9 to 4.7 ± 9.8 pg/ forming cells (SFCs) that specifically responded to the ml), IL-6 (8.2 ± 7.4 to 8 ± 8.6 pg/ml), IL-10 (6.2 ± 4.3 to 5.4 ± 2.7 recombinant S protein after 28 dpi (0 dpi = 129 ± 402, 28 pg/ml), and IL-1b (11.7 ± 8.5 to 11.7 ± 18.8 pg/ml) (Figure 1). dpi = 343.5 ± 1,199) (Figure 4A). In most of the cases, IFN-g SFCs continue to increase at day 90 dpi (90 dpi = 490 ± 384.2). CoronaVac Induces Total and Neutralizing Similarly, a significant increase in the number of granzyme SFCs was also observed in all, but one sample, after 28 dpi (0 dpi = Anti-Spike Antibodies in All Vaccinated 129 ± 402, 28 dpi = 256 ± 152.4) (Figure 4B), and most of them Volunteers increased further at day 90 dpi. Altogether, these results suggest We measured the level of the anti-SARS-CoV-2 spike antibodies the activation and presence of CD4+ Th and cytotoxic CD8+ in 15 volunteers before and after vaccination (Figure 2A). We T memory cells, all required for an effective viral clearance (10). found an increase in the anti-S antibodies in all volunteers after 28 dpi (0 dpi = 24 ± 19, 28 dpi = 759 ± 278); however, after 90 dpi, the anti-S antibody levels declined (90 dpi = 137 ± 92.3), although they were higher than pre-immunization levels DISCUSSION (Figure 2A). When we analyzed data from volunteers for In this study, we analyzed the immune response before immunization, 28 and 90 days after the second immunization TABLE 1 | Demographic and baseline characteristics of enrolled volunteers. (dpi) with CoronaVac during the current COVID-19 mass vaccination schedule applied in Chile. Notably, the results Characteristics indicate that CoronaVac induces a robust humoral immune Male, n (%) 11 (52.3) response and cellular immune memory of at least 90 dpi. Age, mean (range) 36 (27–61) CoronaVac against SARS-CoV-2 triggers the secretion of IL-8 Comorbidities or conditions in all volunteers tested. In PBMC and THP-1 cells, the S protein Obesity (BMI >30), n (%) 2 (9.5) from SARS-CoV also increased IL-8 levels (11). Similarly, Metabolic conditions♠, n (%) 1 (4.8) Hyperlipidemia, n (%) 1 (4.8) SARS-CoV-2 Nsp14 protein, which contributes to the Hypertension, n (%) 1 (4.8) activation of NF-kB signaling, also induces the increase in IL-8 Cardiovascular disease, n (%) 0 and IL-6 (12). This suggests that at least SARS-CoV-2 Spike and Chronic pulmonary disease, n (%) 1 (4.8) Nsp14 proteins can be responsible to produce IL-8 in the Rheumatologic disease, n (%) 1 (4.8) vaccinated volunteers. The impact of sustained higher levels of Immunocompromised, n (%) 0 Allergy♣, n (%) 6 (28.6) IL-8 after vaccination protocol is unclear; however, in ICU Asthma, n (%) 2 (9.5) patients, IL-8 levels are predictive of survival in primo-infected Smoker, n (%) 3 (14.3) patients (13). BMI, Body mass index. ♠Metabolic conditions include insulin resistance and prediabetes. Most of the vaccine’s protection is generated by the ♣ Allergy considered allergic rhinitis, food allergy, and drug allergy. production of specific antibodies; however, the levels, duration, Frontiers in Immunology | www.frontiersin.org 4 January 2022 | Volume 12 | Article 766278 Escobar et al. CoronaVac 28-Day Schedule Vaccination A B C D E F FIGURE 1 | Pro- and anti-inflammatory cytokines in the plasma from CoronaVac-vaccinated volunteers. The levels of (A) IL-8, (B) IL-12, (C) IL-6, (D) IL-1b, (E) IL- 10, and (F) TNF-a were measured by flow cytometry as described in Materials and Methods. Data represented the cytokine level on each volunteer before (Day 0) and 28 days after the second immunization (dpi). The data show individual paired values. Asterisk (**) represents significant difference (p < 0.01). n/s indicates non- significant differences. and protection of circulating antibodies vary depending on the A robust antibody response is insufficient to avoid severe type of pathogen and its immunogenic characteristics (14). Our disease (16) and the cellular immune response is also required for data show that 100% of the volunteers participating in this study an effective viral clearance (10). Here, results show that present seroconversion and neutralizing antibodies production CoronaVac induced a cellular response against the S protein of after 28 and 90 dpi. The induction of neutralizing antibodies after SARS-CoV-2. The increase in IFN-g and Granzyme B-producing CoronaVac administration in a 28-day interval between the first cells after antigen stimulation suggests the activation of CD4+ Th and second dose is consistent with the data reported in the phase cells and cytotoxic CD8+ T memory cells. The presence of T cell- 3 trial of CoronaVac conducted in Chile, which showed over 90% mediated response after CoronaVac vaccination concurs with seroconversion for neutralizing antibodies after 42 dpi (4). BNT162b1 vaccine, which elicited robust CD4+ and CD8+ T-cell Neutralizing antibodies are critical for vaccine efficacy in responses until 43 dpi (17). The ability of CoronaVac to activate preventing infection since robust neutralizing-antibody a cellular immunity is relevant since CD4+ and CD8+ T-cell- responses appear to be more protective than those with weaker mediated response was significantly lower in severe/critical response (15). Although in our current study the neutralizing patients in comparison to those with mild and mid-grade antibodies wane after 3 months of the second dose of groups, data that strongly support the participation of T cells CoronaVac, further studies and characterization of memory B in the immune protection against COVID-19 (18). and plasma cells are needed to associate this condition with a In summary, our analysis showed that two dose potential lack of protection against COVID-19. immunizations with CoronaVac vaccine generates neutralizing Frontiers in Immunology | www.frontiersin.org 5 January 2022 | Volume 12 | Article 766278 Escobar et al. CoronaVac 28-Day Schedule Vaccination A B FIGURE 2 | Total anti-S antibodies in the plasma of CoronaVac-vaccinated volunteers. Anti-S antibodies were quantified in plasma obtained prior to immunization (Day 0), and 28 and 90 days after the second immunization (dpi). (A) Antibody titers are expressed as the area under the ELISA OD curve (AUC) made from four serial dilutions (1/200, 1/400, 1/800, and 1/1600) for each volunteer (▾). The data show individual paired values. (B) Detail of the antibody titers for the volunteers with complete follow-up (n = 15) throughout the study (data from A;○), and those with no sample on some of the time points evaluated (•) were graphed and shown with median. Asterisk (*) represents statistically significant difference compared to baseline (p < 0.05). Hashtag (#) represents statistically significant difference between 28 dpi and 90 dpi samples. n/s, not significant (p < 0.05). A B C D E FIGURE 3 | Total anti-S neutralizing antibodies in the plasma of CoronaVac-vaccinated volunteers. The neutralization curves from plasma samples of volunteers were obtained by using a pseudotyped viral particle (HIV-1–SD19). Samples were titrated in triplicate at serial threefold dilutions and expressed as percent of neutralization. (A) Total anti-S neutralizing antibodies before vaccination (Day 0). (B) Total anti-S neutralizing antibodies at 28 days after the second immunization (dpi). (C) Total anti-S neutralizing antibodies at 90 dpi (D) IC50 calculated based on inhibition rates and depicted for each sample obtained from volunteers with a complete follow-up schedule (□) (n = 15). The data show individual paired values. (E) IC50 calculated for the total data (n = 21 volunteers, including those with no complete follow-up schedule) were graphed and shown with median. Open circles (○): data from (D) Black circles (•): volunteers with no sample on some of the time-points evaluated. Asterisk (*) represents statistically significant difference obtained from the comparison against day 0 (p < 0.05). Hashtag (#) represents statistically significant difference between 28 dpi and 90 dpi samples. n/s, not significant (p < 0.05). Frontiers in Immunology | www.frontiersin.org 6 January 2022 | Volume 12 | Article 766278 Escobar et al. CoronaVac 28-Day Schedule Vaccination A B C FIGURE 4 | Cytokine secreting cells of vaccinated volunteers in vitro induced by SARS-CoV-2 S protein. (A) IFN-g (○). (B) Granzyme (•). The number of ELISPOT- forming cells (SFC) per 3 × 105 was quantitated in PBMC, obtained before vaccination (indicated as "Day 0"). Twenty-eight and 90 days after the second immunization (dpi) (n = 15 volunteers), the data show individual paired values. (C) Total data from 21 volunteers (same volunteers from (A, B) and others with no sample on some of the time points evaluated) were graphed and shown with median for IFN-g (○) and Granzyme (•). Asterisk (*) represents statistically significant difference (p < 0.05) obtained from the comparison against Day 0. n/s indicates non-significant differences. antibodies, IFN-g-producing cells, and Granzyme B 28-days after AUTHOR CONTRIBUTIONS the second immunization. Moreover, the increase in Granzyme B was sustained after 90 days of the second immunization. It is of great ELISPOT and cell isolation: AE, HP, JG, and CB-A. Antibody importance for public health to determine over time the levels of neutralization assays: MA, LA-P, FV-E, and RS-R. Total neutralizing antibodies, cellular immunity, and the dynamic of antibody and cytokine analysis: MB, LV, and VS. S protein memory response to SARS-CoV-2 in the population after production: CV, JH, and LC. Study coordination: DV. MNS: vaccination to determine the need for modifications in the EN-L. MNS and study design. AE, FER-L, AMS, MI, and CA-C. immunization protocols, application of a third/booster dose with All authors contributed to the article and approved the the same or a different vaccine, and improvement offuture vaccines. submitted version. DATA AVAILABILITY STATEMENT FUNDING The raw data supporting the conclusions of this article will be The authors thank the support of ANID-COVID1005 (EN-L), made available by the authors, without undue reservation. ANID-COVID1038 (AMS, FER-L, MI, CA-C), FONDECYT Postdoctoral fellowship N° SECTEI/138/2019 (LA-P), FONDECYT grants 1211547 (FV-E), 1211841 (FER-L), 1201240 ETHICS STATEMENT (CV, JH, and LC), 1201664 (MI, AMS, and FER-L), 1181814 (EL-S), 1190156 (RS-R), and 1180666 (AE); DICYT 021943AC The studies involving human participants were reviewed and (CA-C), SOFOFA SiEMPRE 18 CTBT-102728 (CV, JH, and LC). approved by Ethics Committee, Universidad de Santiago de The funding bodies were not involved in the study design, Chile (172/2021). The patients/participants provided their collection, analysis and interpretation of data, the writing of this written informed consent to participate in this study. article, or the decision to submit results for publication. With the Adsorbed COVID-19 (Inactivated) Vaccine Manufactured by REFERENCES Sinovac – PROFISCOV: A Structured Summary of a. Trials (2020) 21:21–3. doi: 10.1186/s13063-020-04775-4 1. Tregoning JS, Flight KE, Higham SL, Wang Z, Pierce BF. Progress of the 4. Bueno SM, Abarca K, Gonzá lez PA, Gá lvez NMS, Soto JA, Duarte LF, et al. COVID-19 Vaccine Effort: Viruses, Vaccines and Variants Versus Efficacy, Interim Report : Safety and Immunogenicity of an Inactivated Vaccine. Effectiveness and Escape. Nat Rev Immunol (2021) 21(10):626–36. medRxiv (2021). doi: 10.1101/2021.03.31.21254494 doi: 10.1038/s41577-021-00592-1 5. Ministerio de Salud Gobierno de Chile. Ficha Vacuna Contra Sars-Cov-2 2. Mulligan MJ, Lyke KE, Kitchin N, Absalon J, Gurtman A, Lockhart S, et al. Vacuna Coronavac Laboratorio Sinovac Life Science® (2021). Available at: Phase I/II Study of COVID-19 RNA Vaccine BNT162b1 in Adults. Nature https://www.minsal.cl/wp-content/uploads/2021/01/Ficha-vacuna-Sinovac- (2020) 586:589–93. doi: 10.1038/s41586-020-2639-4 Life-Science.pdf (Accessed August 23, 2021). 3. Palacios R, Patiño EG, de Oliveira Piorelli R, Conde MTRP, Batista AP, Zeng 6. Jara A, Undurraga EA, Gonzá lez C, Paredes F, Fontecilla T, Jara G, et al. G, et al. Double-Blind, Randomized, Placebo-Controlled Phase III Clinical Effectiveness of an Inactivated SARS-CoV-2 Vaccine in Chile. N Engl J Med Trial to Evaluate the Efficacy and Safety of Treating Healthcare Professionals (2021) 385:875–84. doi: 10.1056/nejmoa2107715 Frontiers in Immunology | www.frontiersin.org 7 January 2022 | Volume 12 | Article 766278 Escobar et al. CoronaVac 28-Day Schedule Vaccination 7. Huang AT, Garcia-Carreras B, Hitchings MDT, Yang B, Katzelnick LC, From Symptomatic SARS-CoV-2 Infection. Nat Med (2021) 27:1205–11. Rattigan SM, et al. A Systematic Review of Antibody Mediated Immunity doi: 10.1038/s41591-021-01377-8 to Coronaviruses: Kinetics, Correlates of Protection, and Association 16. Vabret N, Britton GJ, Gruber C, Hegde S, Kim J, Kuksin M, et al. Immunology With Severity. Nat Commun (2020) 11:1–16. doi: 10.1038/s41467-020- of COVID-19: Current State of the Science. Immunity (2020) 52:910–41. 18450-4 doi: 10.1016/j.immuni.2020.05.002 8. Beltrá n-Pavez C, Riquelme-Barrios S, Oyarzú n-Arrau A, Gaete-Argel A, 17. Sahin U, Muik A, Derhovanessian E, Vogler I, Kranz LM, Vormehr M, et al. Gonzá lez-Stegmaier R, Cereceda-Solis K, et al. Insights Into Neutralizing COVID-19 Vaccine BNT162b1 Elicits Human Antibody and TH1 T Cell Antibody Responses in Individuals Exposed to SARS-CoV-2 in Chile. Sci Adv Responses. Nature (2020) 586:594–9. doi: 10.1038/s41586-020-2814-7 (2021) 7(7):eabe6855. doi: 10.1126/sciadv.abe6855 18. Yang G, Feng F, Li X, Zhang T, Li X, Li B. Changes of Lymphocyte Subsets in 9. Palgen JL, Tchitchek N, Elhmouzi-Younes J, Delandre S, Namet I, Patients With COVID-19 and Clinical Significance: A Case-Control Rosenbaum P, et al. Prime and Boost Vaccination Elicit a Distinct Innate Observational Study. J Bio-X Res (2021) 4:36–9. doi: 10.1097/jbr. Myeloid Cell Immune Response. Sci Rep (2018) 8:1–18. doi: 10.1038/s41598- 0000000000000089 018-21222-2 10. Channappanavar R, Zhao J. Perlman S. T Cell-Mediated Immune Response to Conflict of Interest: The authors declare that the research was conducted in the Respiratory Coronaviruses. Immunol Res (2014) 59:118–28. doi: 10.1007/ absence of any commercial or financial relationships that could be construed as a s12026-014-8534-z potential conflict of interest. 11. Dosch SF, Mahajan SD, Collins AR. SARS Coronavirus Spike Protein-Induced Innate Immune Response Occurs via Activation of the NF-kb Pathway in Publisher’s Note: All claims expressed in this article are solely those of the authors Human Monocyte Macrophages In Vitro. Virus Res (2009) 142:19–27. and do not necessarily represent those of their affiliated organizations, or those of doi: 10.1016/j.virusres.2009.01.005 the publisher, the editors and the reviewers. Any product that may be evaluated in 12. Li T, Kenney AD, Liu H, Fiches GN, Zhou D, Ayan Biswas J, et al. SARS-CoV- this article, or claim that may be made by its manufacturer, is not guaranteed or 2 Nsp14 Activates NF-kb Signaling and Induces IL-8 Upregulation. BioRxiv endorsed by the publisher. (2021). doi: 10.1101/2021.05.26.445787 13. Li J, Rong L, Cui R, Feng J, Jin Y, Yu Y, et al. Dynamic Changes in Serum Copyright © 2022 Escobar, Reyes-Loṕ ez, Acevedo, Alonso-Palomares, Valiente- IL-6, IL-8, and IL-10 Are Associated With the Outcome of Patients With ́ Soto-Rifo, Portillo, Gatica, Flores, Nova-Lamperti, Barrera-Avalos, Echeverrıa, Severe COVID-19 in ICU. Res Sq (2021) 10(4):3706–14. doi: 10.21203/rs.3.rs- Bono, Vargas, Simon, Leiva-Salcedo, Vial, Hormazabal, Cortes, Valdeś , Sandino, 83336/v1 Imarai and Acuña-Castillo. This is an open-access article distributed under the terms 14. Amanna IJ, Carlson NE, Slifka MK. Duration of Humoral Immunity to of the Creative Commons Attribution License (CC BY). The use, distribution or Common Viral and Vaccine Antigens. N Engl J Med (2007) 357:1903–15. reproduction in other forums is permitted, provided the original author(s) and the doi: 10.1542/peds.2008-2139LLLL copyright owner(s) are credited and that the original publication in this journal is 15. Khoury DS, Cromer D, Reynaldi A, Schlub TE, Wheatley AK, Juno JA, et al. cited, in accordance with accepted academic practice. No use, distribution or Neutralizing Antibody Levels Are Highly Predictive of Immune Protection reproduction is permitted which does not comply with these terms. Frontiers in Immunology | www.frontiersin.org 8 January 2022 | Volume 12 | Article 766278