Booster Vaccination with BNT162b2 Improves Cellular and Humoral Immune Response in the Pediatric Population Immunized with CoronaVac

The SARS-CoV-2 Omicron variant and its sublineages continue to cause COVID-19-associated pediatric hospitalizations, severe disease, and death globally. BNT162b2 and CoronaVac are the main vaccines used in Chile. Much less is known about the Wuhan-Hu-1 strain-based vaccines in the pediatric population compared to adults. Given the worldwide need for booster vaccinations to stimulate the immune response against new Omicron variants of SARS-CoV-2, we characterized the humoral and cellular immune response against Omicron variant BA.1 in a pediatric cohort aged 10 to 16 years who received heterologous vaccination based on two doses of CoronaVac, two doses of CoronaVac (2x) plus one booster dose of BNT162b2 [CoronaVac(2x) + BNT162b2 (1x)], two doses of CoronaVac plus two booster doses of BNT162b2 [CoronaVac(2x) + BNT162b2 (2x)], and three doses of BNT162b2. We observed that the [CoronaVac(2x) + BNT162b2 (2x)] vaccination showed higher anti-S1 and neutralizing antibody titers and CD4 and CD8 T cell immunity specific to the Omicron variant compared to immunization with two doses of CoronaVac alone. Furthermore, from all groups tested, immunity against Omicron was highest in individuals who received three doses of BNT162b2. We conclude that booster vaccination with BNT162b2, compared to two doses of CoronaVac alone, induces a greater protective immunity.


Introduction
Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is a positivestranded ssRNA(+) virus that has led to the severe coronavirus disease 2019 (COVID- 19) pandemic that claimed more than seven million lives worldwide [1].In Chile, a total of 62,249 deaths including confirmed, suspected, and probable cases have been reported, nearly all among adults [2].The clinical manifestations of SARS-CoV-2 primary infections in children and adolescents are in general less severe compared to those in naïve adults.They rarely present severe respiratory symptoms and often remain asymptomatic [3].However, they can develop the life-threatening multisystem inflammatory syndrome MIS-C, while generally lacking severe respiratory symptoms [4][5][6][7].The spike glycoprotein is the dominant exposed antigen on enveloped viruses and hence includes hot spots for mutations occurring in new variants, like Omicron lineages [8].The spike protein includes the S1 subunit spanning the receptor binding domain (RBD), the N-terminal domain (NTD), and the more conserved S2 membrane fusion subunit [9,10], which are important targets for viral neutralization [11].The spike can trigger a series of adaptive immune responses mediated by three major cell types: B cells (humoral immunity) and CD4+ and CD8+ T cells (cell-mediated immunity) [12,13].While the RBD and NTD domains are exposed on the tip of S1 and are the target of most neutralizing antibodies [14], the less exposed S2 spike subunit is more conserved and includes cross-reactive epitopes with seasonal betacoronaviruses [15].Pediatric individuals with previous SARS-CoV-2 infection generally exhibit higher titers of antibodies against the viral spike glycoprotein than adults [16,17].Additionally, children under 5 years of age are found to have a great incidence of infection with the betacoronavirus HCoV-OC43, which is the common cold coronavirus that is most closely related to SARS-CoV-2, which may in part explain the high number of asymptomatic cases [15,18,19].
Wide vaccination coverage is a way to alleviate health systems, and an essential aspect to avoid collapsing medical care and to gain control over a pandemic.For COVID-19, cases continue to increase despite global vaccination campaigns [20,21].Those include numerous safe and effective vaccines that have been developed to effectively reduce the risk of infection, severe disease, and death.Most vaccine strategies employ the spike protein as a target including the spike-coding vaccines based on mRNA BNT162b2 (Pfizer-BioNTech,) NY, USA; Mainz, Germany, mRNA-1273 (Moderna, Cambridge, MA, USA), and adenoviral vectors such as Ad26.COV2.S (Janssen-Johnson & Johnson, Beerse, Belguium; New Brunswick, NJ, USA), among others [22,23].On the other hand, vaccines based on whole-inactivated viruses such as CoronaVac (Sinovac, Beijing, China) also include the spike protein in addition to other viral structural proteins such as the nucleoprotein [24].Several studies have shown that antibody responses from the first massive vaccination against COVID-19 in early 2021 have decreased over the following six months after vaccination, likely contributing to an increase in breakthrough infections [25][26][27].The emergence of numerous SARS-CoV-2 variants is one of the most important developments in the COVID-19 pandemic [28].Yet, diverse variants of concern, interest and under-monitoring have different levels of increased transmissibility and resistance to existing immunity and have emerged sequentially [29].Furthermore, these variants have been widespread and have evolved over time since the start of the pandemic [30][31][32].
To the best of our knowledge, the virological and immunological characteristics associated with the viral main lineages are key to informing health policies, including booster and vaccination schedules, and also informing the development of potential specific variants or pan-coronavirus vaccines.Important issues include whether the different variants escape vaccination-induced immune responses.In adults, mRNA-based vaccines recognize early variants of SARS-CoV-2, while significant overall decreases in humoral and cellular responses to Omicron lineages were recorded [33].In addition, booster doses in adults induce neutralizing immunity against Omicron BA.1 infection [34].A low effectiveness of the BNT162b2 and CoronaVac vaccines against several Omicron variants of SAR-CoV-2 has been described, reporting low levels of neutralizing antibodies in those vaccinated with BNT162b2 and loss of antibody reactivity in those vaccinated with CoronaVac [35,36].
Yet, scarce information on the immune responses in the pediatric population immunized with vaccination schemes based on CoronaVac and heterologous BTN162b2 booster doses is available [37,38].It has been described that vaccination against COVID-19 in children aged 5-11 years showed lower effectiveness in preventing SARS-CoV-2 infection and severe COVID-19 than in individuals aged 12 years and older [39].The CoronaVac vaccine has been associated with weaker neutralization titers against spike but yet comparable or higher T cell responses than mRNA and other vaccine platforms [40].Clinical phase 1/2 studies with CoronaVac have shown that a two-immunization schedule (3 µg dose) induced humoral immune response and neutralizing antibodies in children and adolescents aged 3-17 years [41].T-cell responses are elicited by the inactivated whole-virus vaccines, which are directed against all structural proteins of SARS-CoV-2 and not the spike protein alone.It has been further described that T cell responses against SARS-CoV-2 S, N, and M proteins are conserved for Omicron BA.1 [42].Yet, vaccinated pediatric age groups are more susceptible to reinfections due to Omicron variants than previous variants [43].However, it has been shown that three doses of BNT162b2 or three doses of CoronaVac were effective in preventing COVID-19, hospitalizations, and severe outcomes among the pediatric population during the Omicron-dominant pandemic [44,45].Yet, more studies of pediatric cohorts are required to test their immune status against Omicron lineages.Also, more studies are needed to characterize the efficiency of heterologous vaccination in children and adolescents against SARS-CoV-2 Omicron variants.In this study, we analyzed which vaccination scheme, including CoronaVac (2x) alone, BNT122b2 (3x) alone or their heterologous immunization induces a higher humoral and cellular immune response against SARS-CoV-2 Omicron BA.1 variant in adolescents between 10 and 16 years.Our findings indicate that booster doses using BNT162b2 in pediatric individuals immunized before with CoronaVac (2x) exhibit an enhanced cellular and humoral immune response against Omicron BA.1 compared to CoronaVac (2x) alone.

Study Design and Population
We recruited teenager (10-16 years old) donors immunized with two doses of Coron-aVac [CoronaVac (2x)], two doses of CoronaVac plus one dose of BNT162b2 [CoronaVac (2x) + BNT162b2 (1x)], two doses of CoronaVac plus two of BNT162b2 [CoronaVac (2x) + BNT162b2 (2x)], or three doses of BNT162b2 [(BNT162b2 (3x)].It is important to note that these vaccination schemes were the most common vaccines used in Chile are CoronaVac (Sinovac, Beijing, China) and BNT162b2 (Pfizer, New York, NY, USA; BioNTech, Mainz, Germany), and the most common third booster shot was BNT162b2, in accordance with the booster vaccine policy implemented by the Chilean National Immunization Program (PNI) [46].Despite being freely available, a considerable group of individuals in Chile has refused to receive booster doses.
The sample collection was performed in schools from the Metropolitan Region of Chile during the second semester of the year 2022 when the SARS-CoV-2 Omicron variants BA.1, BA.2, BA.4,BA.5 and BQ.1 were circulating predominantly [47].The inclusion and exclusion criteria were applied to all groups of vaccinated teenagers aged 10 to 16, who must have the complete vaccination schedule against SARS-CoV-2 at least 30 days since their last immunization, with no history of previous SARS-CoV-2 infection.Also, the participants had no symptoms associated with COVID-19.It is important to note that these vaccination schemes were developed against the original strain of SARS-CoV-2 (Wuhan-Hu-1) when administered in Chile.The main inclusion criteria for donors were individuals between 10 and 16 years old, and no history of SARS-CoV-2 infection at least 8 months from the date of sampling.The main exclusion criteria included individuals under 10 or over 17 years old, pregnant women, individuals with morbid obesity, immunosuppression, or symptoms associated with COVID-19.Individuals who had a history of reinfection by COVID-19 were also excluded.
The vaccinated group included individuals who had completed their immunization schedule at least one month before sample collection and no more than 18 months before the collection.A total of 99 donors were included in the study.After applying a documentary filter, 88 donors were analyzed in this study; 18 S1.
Samples were collected with authorization from the local ethics committee, and all participants and their respective parents or guardians provided informed consent by declaring and signing it.

Serum and PBMCs Preparation
Venous blood was used to obtain serum via a vacuum blood collection tube with clot activation and separating gel (BD Vacutainer) as described previously [48].The tube was stored at room temperature for at least 30 min to allow the blood to clot and then centrifuged at 2000 rpm for 10 min at 4 • C; 2 mL of supernatant serum was recovered and liquated into two cryotubes at 1 mL each and stored at −20 • C until further analysis.Then, another two Vacutainer tubes (Heparin/Lithium) were used for the isolation of peripheral blood mononuclear cells (PBMC) by Ficoll gradient.

Generation of rVSV-SARS2-S-BA.1
Recombinant vesicular stomatitis virus carrying the spike protein of SARS-CoV-2 (rVSV-SARS2-S) has been shown to allow for rapid quantification and correlate well in viral neutralization with convalescent serum compared to the authentic SARS-CoV-2 [49][50][51].Given that this virus contains the spike of the ancestral Wuhan-Hu-1 SARS-CoV-2 strain, we prepared rVSV-SARS2-S containing the spike of the Omicron BA.1 variant.Therefore, the SARS-CoV-2 genome sequences corresponding to the sequencing of samples from individuals infected with the Omicron BA.1 variant in different countries were obtained (GISAID (Munich, Germany) accession ID: EPI_ISL_7373598; EPI_ISL_7373061; EPI_ISL_7371749; EPI_ISL_7370181; EPI_ISL_7368223; EPI_ISL_7358093; EPI_ISL_7358079; EPI_ISL_7358076; EPI_ISL_7358069; EPI_ISL_7356256; EPI_ISL_7355546; EPI_ISL_7354594; EPI_ISL_7352907; EPI_ISL_7350365; EPI_ISL_7350042; EPI_ISL_7349764; EPI_ISL_7348417; EPI_ISL_7346862; EPI_ISL_7345322; EPI_ISL_7337515), performing multiple alignments of the open reading frames corresponding to spike glycoprotein in the Clustal W program (Dublin, Ireland) and subsequently, the generation of the consensus sequence with the EMBOSS Cons tool (Hinxton, England) [52] which was compared with the sequence previously described for omicron BA.1 [53] to confirm the characteristic mutations of the variant.The consensus sequence was next synthesized (GenScript, Queenstown, Singapore) and cloned into the VSV-SARS-CoV-2 spike antigenome plasmid [49] (kindly provided by Dr. Kartik Chandran, Albert Einstein College of Medicine, NY, USA), replacing the sequence of the Wuhan-Hu-1 spike.This plasmid also encodes an eGFP reporter gene as an independent transcriptional unit.Recombinant virus rescue was carried out as previously described [49].Supernatants from transfected cells were transferred to Vero E6 hACE2 cells (previously generated [50]) every day until the appearance of eGFP-positive cells examined under an inverted mi-croscope (IX71; Olympus, Tokyo, Japan) and pictures taken (ProgRes C5; Jenoptik, Jena, Germany) for subsequent analyses (Supplementary Figure S1).RNA from the virus in infection passage 9 was sequenced (Macrogen, Singapore) to corroborate that the coding fragment corresponded to the spike of Omicron BA.1, including the additional non-silent mutations K145E y F372S, due to serial passaging.All experiments for the generation of rVSV-SARS2-S-BA.1 and the use of rVSV-SARS2-S viruses in the microneutralization assays were carried out at biosafety level 2.

rVSV-SARS2-S Microneutralization Assays
Microneutralization assays were performed with rVSV-SARS2-S as described previously [50].Briefly, the serum samples were treated at 56 • C for 30 min prior to use.Serial sera dilutions were then incubated with rVSV-SARS2-S-Wuhan-Hu-1 [49], (kindly provided by Dr. Kartik Chandran, Albert Einstein College of Medicine, NY, USA) or rVSV-SARS2-S-BA.1 viruses (MOI of 0.25) for 1 h at 37 • C. The serum-virus inoculum was then added to Vero E6 hACE2 cells and infection was stopped by fixing cells with 4% formaldehyde (Pierce, Waltham, MA, USA) and stained for 5 min with 300 nM 4 ′ ,6diamidino-2-phenylindole (DAPI) (Invitrogen, Waltham, MA, USA).Total fluorescence for GFP was measured on a Synergy plate reader (BioTek, Charlotte, VT, USA) (DAPI excitation at 360 nm and emission at 460 nm; GFP excitation at 485 nm and emission at 526 nm) and normalized against DAPI fluorescence.The half-maximal inhibitory concentration (IC50) of the sera was calculated using non-linear regression analysis based on data obtained from technical replicates.

Statistical Analysis
In this study, the geometric mean and 95% confidence interval (CI) were estimated for each group of immunized individuals.To compare whether there was a statistically significant difference between antibody titers, neutralizing antibodies, and AIM T cells from different vaccine combinations, an ANOVA test was used after the logarithmic transformation of antibody data and Kruskal-Wallis test for AIM T cells [55][56][57][58].To assess the differences in responses between Omicron-BA.1 and Wuhan-Hu-1 within each scheme, a Wilcoxon test was applied.
If a statistically significant result was obtained from the ANOVA test, post hoc pairwise comparisons were performed using Tukey's Honest Significant Difference (HSD) method, which uses the Studentized range distribution to estimate confidence intervals of factor level mean differences.For the Kruskal-Wallis test, Mann-Whitney was applied.
The fold-change (FC) was calculated as the Omicron.BA-1/Wuhan-Hu-1 ratio of T cells in individuals vaccinated with each scheme, and comparisons of the geometric mean values (GMV) of these fold-changes were conducted within and between different vaccination schemes.For this purpose, after logarithmic transformation of the FC, T-test (by T-test compared with a hypothetical mean of 1) and ANOVA with Tukey's post-hoc were applied, respectively.
Next, we analyzed antibodies against S1 (Wuhan-Hu-1).Contrary to the results of antibodies against nucleoprotein, there was a lower titer of antibodies against the S1 protein in the pediatric population immunized with [CoronaVac(2x)] compared with all formulations and boosters based on BNT162b2 (Figure 1B 1B).There were no statistically significant differences between schemes comprising BNT162b2.The data suggest that BNT162b2-based formulations promote an increase in antibodies against S1 in the pediatric population.

• Vaccination schemes based on BNT162b2 improve cellular immune responses against
Omicron BA.1.in pediatric population.
The cellular immune response has previously been described to be of great importance against SARS-CoV-2 infection [61].Despite the large number of spike protein mutations in the Omicron lineages, T-cell recognition is extensively cross-reactive against different SARS-CoV-2 variants [62].We utilized T cell receptor (TCR) dependent AIM assays to identify and quantify spike-specific CD4+ T and CD8+ T cells in the pediatric population.AIM assay is a cytokine-independent method for the identification of Ag-specific T cells based on the increased expression of activation markers after Ag restimulation [63].The T cell responses were analyzed by the activation of CD4+ T lymphocytes through Wuhan-Hu-1 or Omicron BA.1 strain peptide combinations (Figure 3A).The processed data showed that each vaccination scheme presented similar CD4+ T cell activation levels by Wuhan-Hu-1 and Omicron BA.1 spike peptides.In addition, when comparing vaccination schemes, there were no significant differences in activation for Wuhan-Hu-1 and Omicron BA.1.Then, the T cell response was analyzed in the activation of CD8+ T lymphocytes by Wuhan-Hu-1 or Omicron BA.1 variant peptide combinations for the different vaccination schemes (Figure 3B).Within the aforementioned tests, the [CoronaVac (2x)] vaccination scheme with spike peptides from Wuhan-Hu-1 versus Omicron BA.1 was the only one to show significant differences.In this case, Omicron BA.1 spike peptides generated less activation compared to Wuhan-Hu-1 peptides in the scheme based on [CoronaVac(2x)] (p-value = 0.004).the Wuhan Hu-1 or Omicron BA.1 spike proteins.The percentage of AIM of (A) CD4 T cells and (B) CD8 T cells was measured by flow cytometry.To assess the differences in responses between Omicron-BA.1 and Wuhan Hu-1 within each scheme, a Wilcoxon test was applied (* p < 0.05; ns: not statistically significant).A statistically significant difference between groups was determined with one-way Kruskal-Wallis (p < 0.05).Significant pairwise differences were obtained by Mann-Whitney post-hoc (* p < 0.05; ns: not statistically significant).Also, Fold-change (FC), between AIM+ Omicron.BA-1/AIM+ Wuhan (C) CD4 T cells and (D) CD8 T cells in the pediatric population were calculated.The figure displays the geometric mean (GM) for each vaccination scheme, which is represented by a black dot.A statistically significant difference between groups was determined with one-way ANOVA (p < 0.05).Significant pairwise differences were obtained by Tukey's HSD post-hoc test (* p < 0.05; ns: not statistically significant).

Discussion
In this study, we characterized the humoral and cellular immune response against the Omicron variant BA.1 in adolescents attending schools in Santiago, Chile.Our work describes that in adolescents 10-17 years of age, the immunization with [BNT162b2 (3x)] or double BNT162b2 booster vaccination after two doses of CoronaVac [CoronaVac (2x) + BNT162b2 (2x)] promotes humoral immunity and cellular immune response against Omicron BA.1.Neutralizing antibody responses are crucial to reduce COVID-19 disease severity [64].However, significant decreases were observed for memory B cells and neutralizing antibodies over time independently of the SARS-2 variants.In our study, we observed higher IC50 titers in the pediatric cohort compared to adults, which significantly decreased with the Omicron BA.1 strain compared to the ancestral virus.Interestingly, studies have shown that prior infection with SARS-CoV-2 can boost and broaden immunity related to neutralizing antibodies and T cell responses [33].Regarding this point, the pediatric population characterized here had anti-nucleoprotein antibodies, and some individuals showed increased neutralizing antibody titers against Omicron BA.1 compared to the ancestral Wuhan-Hu-1 strain, coinciding with the period of sample collection and suggesting a previous SARS-CoV-2 infection independent from vaccination.Such a possible former infection would be asymptomatic according to the inclusion criteria of our study.Interestingly, children 3-to-5 years of age who had been vaccinated only with two doses of CoronaVac [CoronaVac (2x)] have been shown to be more susceptible to asymptomatic infections with SARS-CoV-2 [65].Hence, in this cohort study, asymptomatic infections may have increased the neutralizing antibody titers independent of vaccination.In any case, even in the presence of possible previous infections, we show that the booster with BNT162b2 promotes anti-spike antibodies, increased neutralization and evokes immunity against Omicron BA.1 in the [CoronaVac (2x) + BNT162b2 (2x)] and [BNT162b2 (3x)] vaccination scheme groups.Such a heterologous prime-boost vaccination with CoronaVac followed by BNT162b2 has also been reported by others to induce high neutralizing titer against SARS-CoV-2 Omicron strains in the pediatric population [66].
SARS-CoV-2 vaccines of different platforms including mRNA, adenoviral vector and inactivated vaccines have been shown to produce T cell responses and have very high effectiveness against hospitalization [67,68].mRNA-based vaccines have been reported to induce memory T lymphocytes that do not affect their response against other variants [33].Furthermore, regardless of the AIM assay, responses against Omicron variants have been described to be highly conserved in CD4+ and CD8+ T cells [33,69].In this context, CoronaVac can stimulate CD4+ T cell responses against spike peptides [69].Our data presented here provide additional evidence that the booster with BNT162b2 generates an increase in the AIM assay of CD4+ and CD8+ T lymphocytes specific to Omicron BA.1 spike protein in the pediatric population immunized with [CoronaVac (2x)].Among the different vaccination groups, we were able to detect significant differences for CD8+ T cell responses only between [CoronaVac (2x)] compared to [BNT162b2 (3x)].However, we did not detect a pronounced T-cell immune enhancement by booster vaccination with BTN162b2 among the other immunization groups as described in previous studies [40,70,71].This difference may be related to the AIM detection method that we used which relies on cytokine-independent measurement of T-cell activation based on a defined peptide pool based on SARS-CoV-2 spike T cell epitopes.Using these peptides, it was that CD4+ T cells in particular show cross-reactivity with spike peptides in 40-60% of SARS-CoV-2 unexposed individuals [56].Such a T cell cross-reactivity may be even more pronounced in our patient cohort involving teenagers, who are at high exposure to common cold coronavirus infections.As a whole, while providing valuable new information, our study also had some limitations.One important limitation concerns our study cohort.At the time when we started recruiting our patient cohort, we were not able anymore to access samples of the pediatric population prior to the SARS-CoV-2 pandemic.Hence, we could not establish a vaccination control group as a baseline to differentiate SARS-COV-2-specific reactivity from cross-reactivity against structural proteins from other coronaviruses, particularly the N protein.By detecting anti-N antibodies, we hence cannot discard that the pediatric cohort may have developed immune responses by asymptomatic infections in addition to the vaccination schemes.This may explain why almost no cell-mediated immunity enhancement effect was observed by booster vaccination with BNT162b2.Further, our cohort showed a variation in the time intervals from the moment of the last booster dose until the sample acquisition among the different groups analyzed.Given the decay of immune responses over time, this variable may have further influenced the obtained results.On the other hand, we could not recruit a CoronaVac group (3x) because it is an uncommon scheme in the Chilean pediatric population.Another limitation of our study is generated by the study design.We only focused on the cellular immune response measured by AIM T cell responses detected only by CD4+ CD134(OX40)+ CD137+ and CD8+ CD69+ CD137+ markers; additional memory T lymphocyte profiles and/or markers such as IL-2 and IFN-γ could provide additional significant information in the comparison the different schemes against spike from Omicron BA.1 vs. Wuhan viruses.Finally, our study was only focused on the entire spike glycoprotein of SARS-CoV-2 and not against S1 and S2 subdomains separately or different epitopes within the RBD.Hence, no information on their neutralizing breadth can be provided as has been established elsewhere [72].
Finally, the findings obtained with our study cohort indicate that vaccination campaigns in the pediatric population implying booster doses, such as BNT162b2 and heterologous schemes, induce enhanced cellular and humoral immune responses against Omicron lineages.Currently, there is apprehension regarding the potential of SARS-CoV-2 emerging variants to evade the immune response elicited by approved vaccines.Vaccine boosters are essential to prevent viral immune evasion and severe forms of disease in the pediatric population.

Informed Consent Statement:
The studies involving human participants were reviewed and approved by Scientific Ethics Committee of the Scientific Ethics Committee from the Southeast Metropolitan Health Service, Santiago, Chile of 19 May 2022.The parents of patients/participants provided their written informed consent to participate in this study.In addition, all the participants signed an informed assent.

Figure 2 .
Figure 2. Neutralizing antibody responses against the ancestral SARS-CoV-2 Wuhan Hu-1 strain and omicron BA.1 variant in pediatric individuals immunized with different schemes of the Coro-naVac and BNT162b2 vaccines.Serum-neutralizing antibody titers were determined by microneutralization assay of recombinant vesicular stomatitis virus carrying SARS-CoV-2 spike protein (rVSV-SARS2-S) from the Wuhan Hu-1 strain or the Omicron BA.1 variant.Data describe statistical analysis based on Log10 [IC50] titers.Dashed line indicates the limit of detection (LOD) of the microneutralization assay.Statistical significance within vaccination schemes was determined with Ttest, and difference between vaccination schemes was determined with one-way ANOVA (p < 0.0001).Significant pairwise differences were obtained by Tukey's HSD post-hoc (* p < 0.05; ns: not statistically significant).

Figure 2 .
Figure 2. Neutralizing antibody responses against the ancestral SARS-CoV-2 Wuhan Hu-1 strain and omicron BA.1 variant in pediatric individuals immunized with different schemes of the CoronaVac and BNT162b2 vaccines.Serum-neutralizing antibody titers were determined by microneutralization assay of recombinant vesicular stomatitis virus carrying SARS-CoV-2 spike protein (rVSV-SARS2-S) from the Wuhan Hu-1 strain or the Omicron BA.1 variant.Data describe statistical analysis based on Log10 [IC50] titers.Dashed line indicates the limit of detection (LOD) of the microneutralization assay.Statistical significance within vaccination schemes was determined with T-test, and difference between vaccination schemes was determined with one-way ANOVA (p < 0.0001).Significant pairwise differences were obtained by Tukey's HSD post-hoc (* p < 0.05; ns: not statistically significant).

Figure 3 .Figure 3 .
Figure 3. CD4+ and CD8+ T-cell responses against SARS-CoV-2 Wuhan Hu-1 strain and Omicron variant in pediatric individuals vaccinated with different schemes of CoronaVac and BNT162b2 vaccines.T cell activation was determined by AIM assay using a megapool of 15-mer peptides resembling the Wuhan Hu-1 or Omicron BA.1 spike proteins.The percentage of AIM of (A) CD4 T cells Figure 3. CD4+ and CD8+ T-cell responses against SARS-CoV-2 Wuhan Hu-1 strain and Omicron variant in pediatric individuals vaccinated with different schemes of CoronaVac and BNT162b2 vaccines.T cell activation was determined by AIM assay using a megapool of 15-mer peptides resembling

Author
Contributions: D.A.D.-D., G.B., D.F.E., P.D. and A.E.V. performed the Collection and processing of samples.D.A.D.-D., G.B., R.P. and N.T.S.-A.assisted in the volunteers' identification, enrollment, collection of epidemiological and clinical data.G.B., P.D. and M.D.-S., performed SARS-CoV-2-specific antibody ELISAs and hACE2 competition assays.S.C.-C., N.A.M. and N.D.T. developed the rVSV-SARS2-S-Omicron-BA.1 virus and performed and analyzed the viral microneutralization assays.DDD performed AIM T cell activation.A.G. and A.S. provided peptide reagents.R.P. and N.T.S.-A.statistical analysis.D.A.D.-D., R.P., N.T.S.-A., S.C.-C., N.A.M., N.D.T. and A.E.V. writing and editing of the manuscript.D.A.D.-D., C.C., J.D., H.E.G.-E.and A.E.V. planning and logistics.All authors have read and agreed to the published version of the manuscript.Funding: This study was financed and supported by the Public Health Institute of Chile.This project has also been funded in part with Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. 75N93021C00016 to A.G. and A.S. and Contract No. 75N93019C00065.NDT received funding from the Agencia Nacional de Investigación y Desarrollo (ANID), Chile through grants FONDECYT 1221811 and Centro Ciencia & Vida, CCTE Basal FB210008.Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki, and approved by Scientific Ethics Committee of the Scientific Ethics Committee (19 May 2022) from the Southeast Metropolitan Health Service, Santiago, Chile.