Immunogenicity and safety of inactivated SARS-CoV-2 vaccine (CoronaVac) using two-dose primary protocol in children and adolescents (Immunita-002, Brazil): A phase IV six-month follow up

Introduction: Vaccines are essential for the prevention and control of several diseases, indeed, monitoring the immune response generated by vaccines is crucial. The immune response generated by vaccination against SARS-CoV-2 in children and adolescents is not well defined regarding to the intensity and medium to long-term duration of a protective immune response, which may point out the need of booster doses and might support the decisions in public health. Objective: The study aims to evaluate the immunogenicity and safety of inactivated SARS-CoV-2 vaccine (CoronaVac) in a two-dose primary protocol in children and adolescent aging from 3 to 17 years old in Brazil. Methods: Participants were invited to participate in the research at two public healthcare centers located in Serrana (São Paulo) and Belo Horizonte (Minas Gerais), Brazil. Participants underwent medical interviews to gather their medical history, including COVID-19 history and medical records. Physical exams were conducted, including weight, blood pressure, temperature, and pulse rate measurements. Blood samples were obtained from the participants before vaccination, 1 month after the first dose, and 1, 3, and 6 months after the second dose and were followed by a virtual platform for monitoring post-vaccination reactions and symptoms of COVID-19. SARS-CoV-2 genome from Swab samples of COVID-19 positive individuals were sequenced by NGS. Total antibodies were measured by ELISA and neutralizing antibodies to B.1 lineage and Omicron variant (BA.1) quantified by PRNT and VNT. The cellular immune response was evaluated by flow cytometry by the quantification of systemic soluble immune mediators. Results: The follow-up of 640 participants showed that the CoronaVac vaccine (Sinovac/Butantan Institute) was able to significantly induce the production of total IgG antibodies to SARS-CoV-2 and the production of neutralizing antibodies to B.1 lineage and Omicron variant. In addition, a robust cellular immune response was observed with wide release of pro-inflammatory and regulatory mediators in the early post-immunization moments. Adverse events recorded so far have been mild and transient except for seven serious adverse events reported on VigiMed. Conclusions: The results indicate a robust and sustained immune response induced by the CoronaVac vaccine in children and adolescents up to six months, providing evidences to support the safety and immunogenicity of this effective immunizer.


INTRODUCTION
The development of safe and effective vaccines to SARS-CoV-2 is crucial for managing the COVID-19 pandemic and will continue to be the primary tool for limiting the virus's spread (GRENFELL et al., 2022).However, the immune response generated by COVID-19 vaccines are still under investigation, and further studies are necessary to gain a better understanding of the protection to SARS-CoV-2 upon vaccination, especially in children and adolescents.In this age group, the immune response triggered by vaccination is not clearly defined in terms of its intensity and duration of protection in the medium and long term, as well as its ability to neutralize distinct variants of concern (VOCs).This information may indicate the need for booster shots and require healthcare managers to make informed decisions (WALTER et al., 2022).
The aim of the present study was to comprehensively assess both humoral and cellular responses and evaluating the effectiveness of the two-dose primary protocol of CoronaVac vaccination in children and adolescents in Brazil.This investigation encompassed the identification of positive COVID-19 cases post-vaccination, infection severity, clinical data, and outcomes in children and adolescents aged 3 to 17 who received the two-dose primary protocol of CoronaVac vaccine (Sinovac/Butantan Institute) against COVID-19 over a six-month period.The data generated throughout this phase 4 monitoring study in children and adolescents will contribute significantly to enhance our understanding of the protective response, effectiveness, and safety of COVID-19 vaccines in this specific age group.

Ethics statement and participants
This study received the approval from the Research Ethics Committee involving Human Subjects at the Oswaldo Cruz Foundation, the Ethics Committee of Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto -University of São Paulo, and the National Council of Ethics in Research (CAAE 55183322.6.0000.5091).Inclusion criteria comprised children and adolescents aged 3 to 17 years, unvaccinated for COVID-19, who, in agreement with their parents or guardians, willingly participated in the study and signed the informed consent and assent forms (ICF/IAF).Informed consent was obtained from the parent(s) and/or legal guardian(s) of all minor participants involved in this study.The consent process included a detailed explanation of the study purpose, procedures, potential risks, and benefits.Participants were informed that their participation was voluntary, and they had the right to withdraw at any time without penalty.Parent(s) and/or legal guardian(s) were provided with contact information for any questions or concerns regarding the study.As for the deferral criteria, participants with suspected SARS-CoV-2 infection waited up to 72 hours for confirmation of the diagnosis.When confirmed, vaccination was postponed for a minimum of four weeks.All methods described in this study were performed in accordance with the relevant guidelines and regulations.

Participant recruitment, sample collection, and follow-up
Participants were invited to participate in the research at two public healthcare centers located in Serrana, São Paulo, and in Belo Horizonte, Minas Gerais, Brazil.Following the acquisition of informed consent and the application of inclusion and deferral criteria, each participant, along with their parents or guardians, underwent an interview with the medical team to obtain a comprehensive medical history, any previous history of COVID-19 infections, and information regarding concurrent medications, whether related or unrelated to their medical history.
After, physical examinations were conducted, encompassing measurements of weight and vital signs such as systolic and diastolic blood pressure, axillary temperature, and pulse rate.After the completion of these examinations, the participants underwent peripheral blood sampling and were then guided through the vaccination procedure within the healthcare facility.
A total of 640 participants who met the inclusion criteria were followed for a duration of six months after completing the two-dose primary protocol of CoronaVac vaccine (Sinovac, Butantan Institute), administered with a 28-day interval between doses.
Peripheral blood samples were collected at multiple time points: prior to vaccination, on the day of the second dose administration, and at one month, three months, and six months post-second dose, with reference to the date of administering the second dose of the CoronaVac vaccine (Sinovac, Butantan Institute), as well as carrying out the physical examinations described previously.One sample of 10 ml of whole blood were obtained via venous puncture from each participant according to biosafety standards and subsequently centrifuged at 3,000 g/5 min to obtain serum for immunogenicity analyses.
All participants were daily monitored through a virtual platform for a duration of seven days to report any post-vaccination adverse events, and continuously for the reporting of

Assessment of anti-S and anti-N IgG antibodies and viral neutralization assays to SARS-CoV-2
All serum samples obtained from individuals participating in the study were subjected to testing for specific total IgG antibodies to Spike (S) and Nucleocapsid (N) proteins of SARS-CoV-2 at all study sample collection times.These proteins, used as antigens, were generated in stable recombinant HEK293 cells, as outlined in the work of ALVIM et al.For the assessment of SARS-CoV-2 neutralizing antibodies, a subset of participants who had not previously been diagnosed with COVID-19 was evaluated at multiple time points: before vaccination, and at one month, three months, and six months post-second dose of the CoronaVac vaccine.Two distinct assays were employed for this purpose: the plaque reduction neutralization test (PRNT) assessed neutralizing antibodies to the SARS-CoV-2 (B.1), while the viral microneutralization assay (VNT50) evaluated neutralizing antibodies against the Omicron variant (BA.1).These assessments were conducted following the protocols outlined by GRENFELL et al. (2022) andCAMPOS et al. (2022).

Identification of adverse events and serious adverse events following vaccine administration
All participants underwent a seven-day monitoring following the administration of each dose of the COVID-19 vaccine, which was facilitated through telephone communication, with the aim of identifying any adverse events (AEs) and serious adverse events (SAEs).
In this study, AEs encompassed any unfavorable medical incidents reported by vaccinated participants, which may not necessarily have had a direct causal connection with vaccine administration.In contrast, SAEs were defined as any adverse event leading to one of the following outcomes: death, risk of death at the time of the event, hospitalization or extension of existing hospitalization, significant or persistent disability that significantly disrupted an individual's ability to carry out routine functions, or clinically significant events arising from the use of necessary medications during a medical intervention aimed at preventing death, significant disability, or hospitalization of the participant.
During in-person follow-up visits and within the initial seven days following each vaccine dose, participants were queried about the occurrence of specific signs and symptoms (solicited AEs), as well as the spontaneous reporting of additional signs and symptoms (unsolicited AEs).The intensity of solicited AEs was categorized using a numerical scale ranging from 1 to 4, as detailed in Supplementary table 1 (local events) and Supplementary table 2 (systemic events), developed in accordance with the Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials by the United States Food and Drug Administration (FDA, USA) and the Common Terminology Criteria for Adverse Events -Version 5.0 by the United States National Cancer Institute (NCI/NIH, USA).Similarly, unsolicited AEs were graded based on this numerical scale, as per Supplementary table 3, in line with the same guidelines.The highest reported intensity for an AE, until its resolution or outcome, was employed for study analyses.Furthermore, AEs were classified based on their causal relationship with the CoronaVac vaccine, following the adapted classification from the Uppsala Monitoring Centre of the World Health Organization (UMC, WHO), as outlined in Supplementary table 4. All local reactions following vaccination were considered AEs with a definitive causal connection to the vaccine.All solicited and unsolicited AEs identified during the initial two weeks after each vaccine dose's administration were documented, irrespective of their causal association with the vaccine.These identified events were meticulously recorded in a spreadsheet utilizing the RedCap software, with immediate notification of SAEs to the VigiMed platform of the Brazilian National Health Surveillance Agency (Anvisa, Brazil).

CoV-2 sequencing by NGS
In case of suspected COVID-19 during the study period, purified RNA from nasopharyngeal swab samples of individuals was used for RT-qPCR using the Charité/Berlin and Centers for Disease Control and Prevention (CDC, USA) protocols (CORMAN, V. et al., 2019).SARS-CoV-2 positive samples with a cycle threshold (Ct) value < 34 were subjected to NGS sequencing, following the protocol described by GRENFELL et al. (2022) andDEZORDI et al. (2022).The obtained sequences were promptly deposited in the Global Initiative on Sharing All Influenza Databank (GISAID).

Statistical analysis and data mining
Data analyses were conducted employing GraphPad Prism® software.The results obtained from antibody titer quantification were subjected to statistical analysis utilizing the Kruskal-Wallis test and Mann-Whitney normality tests, with a significance level set at p < 0.05.Signatures of soluble mediators were derived by transforming serum levels, initially expressed as continuous variables (pg/mL).For the clinical monitoring records, the platforms Cytoscape and RedCap were employed.

Demographic data of the patients
A total of 640 participants ranged in age from 3 to 17 years old (median 9 years) were consented and enrolled in the study.Samples were collected from March 2022 to July 2023.A total of 325 (50.75%) of the participants were male and 315 (49.22%) were female.The most common pre-existing comorbidities were rhinitis 73 (11.41%), asthma 42 (6.56%),obesity 18 (2.81%),sinusitis 9 (1.41%), bronchiolitis 8 (1.25%), and bronchitis 8 (1.25%), as described in Table 1.When evaluating the kinetics of total IgG antibodies against S and N proteins of SARS-CoV-2 (Figure 2), a significant difference in the titers of anti-S (p < 0.0001) and anti-N IgG antibodies (p < 0.0001) was observed one month after completing the primary immunization, and this difference persisted throughout the analyzed period of up to six months for both specific antibodies assessed.Seropositivity rates were 96.02%, 90.08%, and 94.61%, respectively, at 1, 3, and 6 months, when considering antibodies against the S protein of the virus.A similar profile was identified for the nucleocapsid protein, where seropositivity rates were 98.01%, 92.80%, and 92.95%, respectively, at 1, 3, and 6 months after receiving both doses of the CoronaVac vaccine.In the age group (3-5, 6-11, and 12-17 years old) analysis, the kinetics of total IgG antibodies to S and N proteins had the same response profile, with a statistically significant increase in total antibodies one month after the application of the second dose of the CoronaVac vaccine compared to pre-vaccination levels (Figure 3).The levels of neutralizing antibodies determined by the PRNT assay using the B.1 lineage of SARS-CoV-2 (Figure 4A) demonstrated a significant increase in seropositivity rate was observed from 30.35% to 63.41% at 1 month, 87.17% at 3 months, and 95.24% at 6 months (p < 0.0001) after the administration of primary protocol.The mean assessment showed an increase of 108.93% at 1 month, 187.23% at 3 months, and 213.87% at 6 months after vaccination, compared to the initial time point of this study.
Regarding the titers of neutralizing antibodies determined by the VNT50 methodology with Omicron variant (BA.1) (Figure 4B), a seropositivity rate of 71.66% increased to 85% at 1 month, with a significant increase to 95% at 3 months (p = 0.0207), and 97.5% at 6 months after the application of the second dose of the CoronaVac vaccine.The mean assessment showed an increase of 18.61% at 1 month, 32.55% at 3 months, and 36.02% at 6 months after vaccination, compared to the first follow-up time.Regarding the evaluation of neutralizing antibodies against the Omicron variant (BA.1) in these same age groups (Figure 5, C and D), it was observed that only children exhibited a statistically significant difference in the pre-vaccination period compared to the three months post the administration of the second dose of CoronaVac (p = 0.0069).

Serum soluble mediators from participants who had no previous diagnosis of COVID-19
at pre-vaccination or during the 6-months follow up revealed an increase in the levels of chemokines (CCL11, CCL3, CCL4, CCL2, CCL5 and CCL10), pro-inflammatory cytokines (IL-1, TNF-, IL-12, IFN- and IL-15), regulatory cytokines (IL-1Ra, IL-4, IL-5, IL-9, IL-10 and IL-13), and growth factors (FGF-basic, PDGF, VEGF, G-CSF, IL-7 and IL-2) shortly after the first month compared to the pre-vaccination time point (Figure 6).Furthermore, most of the analyzed biomarkers showed a significant increase between 1 and 6 months, except for the pro-inflammatory cytokine IL-12, which exhibited a decrease between the third and sixth month of the study follow-up.When analyzing the soluble serum biomarkers separately by age group, a similar profile was observed among children (Figure 7) in comparison to the overall assessment of study participants, except for the biomarkers CXCL10 and IL-6, which did not show a statistically significant difference during the evaluated follow-up period.As for adolescents (Figure 8), apart from the inflammatory cytokine IL-6, there was no statistically significant difference in the growth factor PDGF between any of the postvaccination time points.Hence, it is evident that a robust and enduring cellular immune response, characterized by a combination of pro-inflammatory and regulatory elements, persists for up to six months after the administration of two doses of the CoronaVac vaccine in the children and adolescents observed within the scope of this study.

Safety Response -Identified AEs and SAEs
One hundred and ninety-two participants (192, 30%) presented AEs, resulting in a total of three hundred seventy-nine (379) events, with three of them occurring in infants (Table 2).Among these, 39.3% (149) were described as solicited systemic adverse events, 33% (125) were unsolicited adverse events, and 26.6% (101) were solicited local adverse events.Among the major adverse events reported by study participants, 24% (91) had pain at the vaccine administration site, 8.7% (33) had fever, and 4.2% (16) had a runny nose, with the majority of these being of intensity grade 1 (69.9%,265).
The recorded adverse events were mild and transient, except for seven Serious Adverse Events (SAEs) (Table 3), which were monitored by the study's clinical team until resolution.Among the SAEs, three were unlikely to be related to the vaccine, and four were classified as not related.All participants had a favorable recovery outcome.4 and 5).Despite a reduction in the overall mean antibody titers observed at three months postvaccination in our study, these averages remained consistently high (above the detection limit of the reference assay) throughout the study period, demonstrating the durability and persistence of the total antibody response.The seropositivity rate reached 90% for anti-S IgG at the third month and 95% after six months of follow-up.Similarly, seropositivity rates reached 93% and 99% for anti-N IgG at the third-and sixth-months postvaccination.Evaluation of the geometric mean titers of anti-S and anti-N IgG antibodies among participants grouped by age revealed a consistent pattern of seropositivity, confirming a substantial increase in total antibodies against SARS-CoV-2 within the first month after vaccination, followed by a stable immune response over the course of the sixmonth study.
antibodies before vaccination, to both for Omicron and the ancestral B lineage, indicating previous, likely asymptomatic exposure.A meta-analysis study published in the Journal of Medical Virology compiled data on asymptomatic infections, revealing that during waves of Omicron variant infections, children and adolescents had a high proportion of asymptomatic cases (82%), while in the elderly (already widely vaccinated at that time), this proportion was lower (62%).These data reinforce the importance of vaccination among children and adolescents, as they may contribute to the ongoing circulation of SARS-CoV-2 (YU et al., 2022).
Regarding the cellular response data demonstrated in this study, in general, a similar profile was observed among the age groups, with occasional differences in proinflammatory cytokines when comparing the profiles of children to adolescents from the third to the sixth month of follow-up.An initial inflammation observed triggered by vaccination is a premise for the development of a robust cellular response.It is noticeable that there was an increasing release of soluble pro-inflammatory and regulatory mediators in the first three months post-vaccination, with the maintenance of the response at six months of post-vaccination follow-up.
Promising results regarding the cellular immune response in children and adolescents who received the CoronaVac vaccine have also been reported, as described by Soto and collaborators (2022).Their findings highlighted a significant increase in CD4 + AIM + Tcells in response to the structural proteins of the virus.In the 12 to 17-year-old group, there was a notable expansion in the activation of CD4 + T-cells in response to all the structural proteins of SARS-CoV-2, indicating that the CoronaVac, as a inactivated virus vaccine, stimulates cellular immunity not only against the Spike (S) protein but also against the membrane (M) and nucleocapsid (N) proteins.Furthermore, an increase in IL-2 secretion in response to the S and N proteins was observed in both age groups, with this inducing the production of neutralizing antibodies and activation of CD4 + T-cells to SARS-CoV-2 variants (SOTO et al., 2022). Recently any suspected COVID-19 symptoms.Those displaying symptoms suggestive of the disease underwent a medical evaluation, which included nasopharyngeal swab collection to confirm the diagnosis via RT-qPCR.Positive samples from these individuals underwent next-generation sequencing (NGS) for further analysis.Data regarding participants' hospitalizations or adverse events were obtained from medical reports and/or the participants' medical records.The study design is illustrated in the schematic representation of Figure 1.

(
2020).The detection of antibodies was carried out using standardized ELISA assays, in accordance with the methodology established by GRENFELL et al. (2022), which had been validated by the National Institute of Health Quality Control of Oswaldo Cruz Foundation (INCQS/Fiocruz).

Figure 2 .
Figure 2. Kinetics of anti-S IgG (A) and anti-N IgG (B) levels of SARS-CoV-2 at prevaccination, 1 month after the first dose, and 1 month, 3 months, and 6 months after receiving the second dose of the CoronaVac vaccine (Sinovac/Butantan Institute).The detection limit of 0.1508 in (A) and 0.1460 in (B) is represented by dashed lines.The black dots represent individual data points of optical density (450nm) for each vaccinated participant.The percentage values indicate the seropositivity rate.The geometric mean of IgG anti-S and anti-N antibody titers is represented by blue and green bars, respectively.Statistical differences defined by Kruskal-Wallis and Mann-Whitney methods are represented for comparisons over time.

Figure 3 .
Figure 3. Kinetics of total IgG anti-S (A) and anti-N (B) antibody levels to SARS-CoV-2 at pre-vaccination, 1 month after the first dose, and 1 month, 3 months, and 6 months

Figure 4 .
Figure 4. (A) Neutralizing antibodies detected by PRNT to B.1 lineage of SARS-CoV-2 in children and adolescents vaccinated with CoronaVac (Sinovac/Butantan Institute).The cutoff for seropositivity definition of 20 is represented by dashed lines.The geometric mean antibody titer is represented by orange bars.The colored points represent the individual result of each participant at different follow-up times in the study.Statistical differences defined by Mann-Whitney are presented for comparisons over time.(B) Neutralizing antibodies detected by VNT50 to Omicron variant (BA.1) of SARS-CoV-2 in children and adolescents vaccinated with CoronaVac (Sinovac/Butantan Institute).The cutoff for seropositivity definition of 20 is represented by dashed lines.The geometric mean antibody titer is represented by red bars.The colored points represent the individual result of each participant at different follow-up times in the study.Statistical differences defined by Mann-Whitney are presented for comparisons over time.

Figure 5 .
Figure 5. (A) Neutralizing antibodies detected by PRNT to B.1 lineage of SARS-CoV-2 in children aged 3 to 11 years vaccinated with CoronaVac (Sinovac/Butantan Institute).The cutoff for seropositivity definition of 20 is represented by dashed lines.The geometric mean antibody titer is represented by orange bars.The colored points represent the individual result of each participant at different follow-up times in the study.Statistical differences defined by Mann-Whitney are presented for comparisons over time.(B) Neutralizing antibodies detected by PRNT to B.1 lineage of SARS-CoV-2 in adolescents aged 12 to 17 years vaccinated with CoronaVac (Sinovac/Butantan Institute).

Table 1 .
Demographic data of participants in the study.

Table 2 .
Adverse events reported after the administration of the CoronaVac vaccine in children and adolescents during a six-month follow-up.

Table 3 .
Serious adverse events reported after the administration of the CoronaVac vaccine in children and adolescents during a six-month follow-up.

Table 4 .
Data on infections, hospitalizations, and deaths among study participants.

Table 5 .
Fernandes et al. (2021)CoV-2 variant obtained by NGS among study participants.Furthermore, a study that assessed the circulation of SARS-CoV-2 in children in Brazil from April 2020 to July 2022 reported a higher risk of infection in children from symptomatic family adults, usually the mother, reinforcing the importance of vaccination across all age groups(FULLER et al., 2023).Still, a comprehensive statistical analysis of the collective data indicated a significant rise in titers of anti-S IgG antibodies and anti-N IgG antibodies, as well as in seropositivity rates, one month after completing the primary immunization course with two doses of the CoronaVac vaccine.Similar findings were reported byFernandes et al. (2021), wherein all children in the study exhibited a substantial increase in antibody titers induced by the CoronaVac vaccine one month after vaccination, underscoring a robust serological response to a single dose of CoronaVac in a pediatric population up to five years old, with no reports of severe adverse effects.