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HEALTH SCIENCES • An. Acad. Bras. Ciênc. 98 (3) • 2026 • https://doi.org/10.1590/0001-3765202620251157 linkcopy

Open-access Immunological and Hepatic Responses to ChAdOx1-S/nCoV-19 Vaccination in Brazilian Academic Volunteers

AuthorshipSCIMAGO INSTITUTIONS RANKINGS

Abstract

COVID-19 is caused by the SARS-CoV-2 coronavirus. Vaccine development was essential for reducing hospitalizations and mortality during the pandemic. In Brazil, the ChAdOx1-S/nCoV-19 (AstraZeneca) vaccine was widely administered during the initial phase of the national immunization campaign. Despite a well-established safety profile, continuous surveillance of adverse events remains important. Mild adverse reactions occurred more frequently after the first dose and were generally self-limiting. The vaccine induced a strong IgG antibody response, demonstrating robust immunogenicity while producing minimal changes in liver function parameters. These findings support the favorable safety, tolerability, and immunogenic profile of the ChAdOx1-S/nCoV-19 vaccine in this population.

Key words
Alanine Aminotransferase; Aspartate Aminotransferase; AstraZeneca Vaccine; Bilirubin; Immunoglobulin; Interleucin-6

INTRODUCTION

The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, has led to alarming global rates of morbidity and mortality. In Brazil, more than 38 million confirmed cases and over 713,000 deaths were reported by September 2024, with approximately 521 million vaccine doses administered by that time (WHO 2023, Long et al. 2022). Vaccination emerged as one of the most effective strategies to reduce the disease severity and transmission by promoting herd immunity (Tregoning et al. 2021). In response to the urgent global need for immunization, around forty different vaccine formulations were developed, with ten approved for emergency or full use by the World Health Organization. The most widely used vaccines include Pfizer–BioNTech (BNT162b2), Oxford–AstraZeneca (ChAdOx1-S/nCoV-19/AZD1222), and Moderna (mRNA-1273) (Khandker et al. 2021). In Brazil, the primary vaccines administered have been CoronaVac (Sinovac Biotech), ChAdOx1, BNT162b2 (Pfizer–BioNTech), and Ad26.COV2.S (Johnson & Johnson–Janssen). These vaccines have significantly reduced the risk of hospitalization, intensive care unit (ICU) admission, and death (Khandker et al. 2021).

Despite the rapid development and deployment of COVID-19 vaccines, reports of severe adverse effects remain rare, supporting their general safety and tolerability (Sharif et al. 2021, Efe et al. 2022). However, many vaccines were authorized without extensive long-term studies regarding their potential side effects. In Brazil, vaccination began mainly with the adenoviral vector-based vaccine ChAdOx1-S/nCoV-19 (AZD1222) (Folegatti et al. 2020). This vaccine is based on a replication-deficient simian adenovirus vector encoding the full-length spike glycoprotein of SARS-CoV-2. Following phase 1 trials in the UK (COV001), additional randomized controlled trials were conducted in the UK (COV002), Brazil (COV003), and South Africa (COV005), demonstrating acceptable safety, as well as the induction of binding and neutralizing antibodies and interferon-γ (IFN-γ) responses, particularly after the second dose (Voysey et al. 2021a).

Although ChAdOx1-S/nCoV-19 has shown a favorable safety profile, rare post-vaccination events have been reported, such as myocarditis (Montgomery et al. 2021), vaccine-induced immune thrombotic thrombocytopenia (VITT) (Arepally & Ortel 2021), IgA vasculitis (Badier et al. 2021), and autoimmune reactions (Zheng et al. 2022). In addition, studies have described cases of liver injury following COVID-19 vaccination, with some resembling autoimmune hepatitis (Bril et al. 2021, Efe et al. 2022). These cases were generally mild and responsive to corticosteroids (Shroff et al. 2022, Cao et al. 2022). Recent investigations have focused on the potential hepatic effects of COVID-19 vaccines, analyzing biomarkers such as interleukin-6 (IL-6), a key cytokine involved in inflammation and immune regulation (Bergamaschi et al. 2021, Rammohan et al. 2023). IL-6 plays an essential role in the development and maintenance of adaptive immune responses and is associated with liver inflammation and injury (Gubernatorova et al. 2020, Li et al. 2022). Furthermore, some studies suggest that vaccination may result in elevated liver enzymes followed by a subsequent decline, indicating temporary liver stress or inflammation (Rammohan et al. 2023).

Therefore, the objective of this study was to evaluate antibody production, IL-6 levels, and the levels of bilirubin and liver enzymes in academic volunteers before and after receiving the ChAdOx1-S/nCoV-19 vaccine. These findings aim to contribute to a better understanding of the vaccine’s immunogenic and hepatic effects.

MATERIALS AND METHODS

Experimental Ethical Criteria

A total of 69 academic and non-academic volunteers who received the SARS-CoV-2 vaccination between March 2021 and June 2022 were recruited during the vaccination campaign. Twenty-seven volunteers who received two doses of the ChAdOx1-S/nCoV-19 vaccine participated in the study. The project was approved by the Ethics Committee of the Universidade Federal do Rio de Janeiro – Campus Macaé (CEP/UFRJ-Macaé; protocol CAAE: 57373422.8.0000.5699). Inclusion criteria comprised receipt of two doses of the ChAdOx1-S/nCoV-19 vaccine within the study period and completion of all scheduled blood collections. Volunteers who did not receive this vaccine, became infected with SARS-CoV-2 during the study, were pregnant, under 18 years old, or unable to complete the sampling protocol were excluded. SARS-CoV-2 infection was monitored weekly by RT-qPCR.

Nasopharyngeal swab collection, RNA extraction, and RT-qPCR

Nasopharyngeal swab samples were collected prior to blood sampling, as detailed in the Biological Specimen Collection Schedule section. Samples were immersed in 2 mL of Dulbecco’s Modified Eagle Medium (DMEM; Gibco, cat. no. 11054020) and labeled. Nucleic acid extraction was performed using MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s instructions. Briefly, 200 μL of the sample suspension in DMEM was used for the extraction procedure. Purified nucleic acids were eluted in 50 μL of RNase-free water. Real-time reverse transcription polymerase chain reaction (RT-qPCR) for SARS-CoV-2 detection was performed using primers and probes as previously described by the Berlin protocol (RdRP and E targets) (Corman et al. 2020) or the CDC protocol (N1 and N2 targets) (CDC 2021). Reverse transcription and amplification reaction were conducted using the TaqMan™ Fast Virus 1-Step Master Mix (Thermo Fisher Scientific).

Volunteer Clinical Data

In addition to signing the ICF, participants completed a questionnaire to provide demographic and health-related information, including comorbidities, medication use, vaccination schedule, and post-vaccination adverse effects.

Biological Specimen Collection Schedule

For biochemical and immunological analyses, blood samples were collected 7 days before the first vaccine dose and at 7, 15, and 30 days after the first dose. Subsequently, samples were obtained 7 days before the second dose and at 7, 15, and 30 days thereafter. Monthly follow-up collections were performed until the third dose (60, 90, and 120 days after the second dose; see Supplementary Material – Figure S1).

S-UFRJ ELISA for the Detection of Anti-S Immunoglobulins

Peripheral blood was collected in 4 mL EDTA K3 tubes (Firstlab). Plasma was separated by centrifugation at 4000 rpm (Daiki DT-5000) for 10 minutes at 25 °C and stored at -20 °C, or -80 °C for long-term preservation. Samples were aliquoted and thawed immediately before the experiments to avoid repeated freeze–thaw cycles. The in-house S-UFRJ ELISA was performed by immobilizing the trimeric SARS-CoV-2 spike protein on high-binding plates, as described by Alvim et al. (2022). Plasma samples were added to the plates, followed by incubation with secondary antibodies at a 1:200 dilution: anti-human IgM (Fc) HRP-conjugated (#MFCD00162459; Sigma), anti-human IgG (H+L) HRP-conjugated (#W4031; Promega), and anti-human IgA HRP-conjugated (#A18781; Invitrogen). Plates were incubated for 1 hour at room temperature. Absorbance was measured at 450 nm using a Multiskan Sky microplate reader (Thermo Fisher Scientific). Results were expressed as optical density units (OD) or as the ratio of sample OD to the cut-off value. The pre-pandemic healthy volunteers (blood samples collected in January 2020) served as negative controls. Following optimization by means of a receiver operatoring characteristic (ROC) analysis, the cut-off was defined as the mean OD of negative controls on the same plate plus three times the standard deviation determined from 90 negative controls, as described by Sousa et al. (2023).

Biochemical and Inflammatory Liver Parameters

Liver enzyme and bilirubin levels were analyzed in plasma samples collected before and after each vaccine dose. Alanine aminotransferase (ALT; MS80022230086; Gold Analisa), aspartate aminotransferase (AST; MS80022230083; Gold Analisa), and total/direct/indirect bilirubin (MS80022230140; Gold Analisa) were quantified using colorimetric methods. ALT and AST activities were measured using the UV kinetic method (Reitman & Frankel 1957) at 340 nm and 25 °C. Bilirubin levels were determined using the Sims-Horn colorimetric method (Sims & Horn 1958) at 525 nm and 24 °C. Enzyme levels were reported in U/L and bilirubin in mg/dL. IL-6 was measured using a human ELISA kit (#RAB0306-1KT; Sigma) and expressed in pg/mL based on a standard curve.

Variable Selection and Principal Component Analysis

Principal Component Analysis (PCA) was performed including IgG, IgM, IgA, ALT/AST ratio, total bilirubin, IL-6, and time points (0, 7, 15, and 30 days) to evaluate the collective contribution of these variables to the overall response pattern. To address potential multicollinearity among immunological and biochemical parameters, PCA was conducted using a correlation matrix. Dissimilarities between observations were calculated using the Euclidean distance metric. The analysis was carried out using PAST software (version 4.03) (Hammer et al. 2001).

Statistical Analysis

Statistical analyses were performed using GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA). The Shapiro-Wilk tests were used to assess data normality. Comparisons between two independent groups were analyzed using the Mann-Whitney U test. The Wilcoxon signed-rank test was used to compare paired samples. For comparisons across multiple groups, the Kruskal-Wallis test followed by Dunn’s post-hoc test was applied. A p-value < 0.05 was considered statistically significant. Data are expressed as the median, minimum, and maximum.

RESULTS

Between March 2021 and June 2022, 69 academic volunteers were initially recruited for the study. Participants were excluded from the analysis due to SARS-CoV-2 infection (monitored weekly by RT-qPCR) or missing blood samples at scheduled time points. Consequently, 13 volunteers met the eligibility criteria for inclusion in the final analysis (Figure 1). Among these participants, nine (69.2%) were women and four (30.8%) were men, with ages ranging from 26 to 55 years. The median age was 35 years for women and 45.5 years for men. The cohort was highly educated, comprising undergraduate students, graduate students, and university faculty. Regarding comorbidities, five participants (38.5%) were obese, two (15.4%) had hypothyroidism, and one (7.7%) presented with other chronic conditions. Additionally, one participant reported the use of immunosuppressive medication, and two (15.4%) used levothyroxine, commonly prescribed for hypothyroidism (Table I).

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Table I
Sociodemography characteristics of the cohort: age, Body Mass Index (BMI), comorbidities, and medication. (n= 13).
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Figure 1
Eschematic representation of volunteers selection, vaccine distribution, and follow-up process. A total of 69 volunteers provided informed consent. Of these, 37 completed both the first and second doses of a COVID-19 vaccine: BNT162b2 (n = 2), CoronaVac (n = 8), and ChAdOx1-S/nCoV-19 (n = 27). Among those vaccinated with ChAdOx1-S/nCoV-19, 18 completed the second dose. Nine participants were excluded due to SARS-CoV-2 infection or loss to follow-up. An additional five were excluded due to incomplete data, resulting in a final analytical cohort of 13 participants.

The volunteers reported the occurrence of adverse reactions around one day post-vaccination, ten (77%) volunteers reported experiencing some adverse reactions after the first dose of the vaccine and seven (61.5%) reported adverse reactions after the second dose of the vaccine. The highest adverse reactions after the first dose were fever (n=9, 69%), headache (n=9, 69%), body ache (n=7, 54%), fatigue (n=7, 54%) and local pain (n=7, 54%; Figure 2a). In contrast, after the second dose of the vaccine, no adverse reactions were reported in six (46%) of the volunteers. The remaining volunteers reported fever (n= 4, 31%), local pain (n= 4, 31%), and headache (n= 4, 31%) (Figure 2b).

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Figure 2
Adverse effects related by volunteers after ChAdOx1-S/nCoV-19 vaccination. (n= 13) (a) Symptoms after the first dose. (b) Symptoms after the second dose.

The production of Anti-S SARS-CoV2 antibodies (IgM, IgA, and IgG) was monitored until 30 days after the vaccination by ChAdOx1-S/nCoV-19 (Figures 3 and 4). The blood collection was performed before the first dose and 7, 15, and 30 days after the first and second dose of vaccination. Plasma IgM, IgA and IgG levels did not change during the first week after the first vaccine dose (Figure 3a, b, c). IgM and IgA levels remained low between 7 and 15 days after the first dose; however, IgG levels increased 2.3-fold from day 7 (median 0.5540, range 0.4200-0.8010 OD) to day15 (median 1.266, range 0.6920-2.313 OD) (Figure 3d-f). Analysis from 15 to 30 days after the first vaccine dose revealed that the IgM levels from only two volunteers were positive and only one volunteer were positive for IgA, despite all volunteers showing an elevation of IgA levels even under the cut-off value. IgM production remained positive in only one volunteer at 30 days, and IgA levels increased in only one volunteer between 15 and 30 days (Figure 3g, h). IgG levels increased 1.6-fold from day 15 (median 1.266, range 0.6920-2.313 OD) to day 30 (median 2.069, range 0.9000-3.110 OD) after the first dose (Figure 3i).

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Figure 3
Levels of IgM, IgA, and IgG production after the first dose of the ChAdOx1-S/nCoV-19 vaccine. (n= 13) (a-c) Comparison of immunoglobulin production before and 7 days after vaccination. (d-f) Comparison of immunoglobulin production 7 and 15 days after vaccination. (g-i) Comparison of immunoglobulin production 15 and 30 days after vaccination. The round symbols represent women, and the square symbols represent men. The dashed line indicates cut-off value. ****p < 0.0001 represents the difference between 7 and 15 days after the first ChAdOx1-S/nCoV-19 dose; *p < 0.05 represents the difference between 15 and 30 days after the first ChAdOx1-S/nCoV-19 dose.

After the second dose of ChAdOx1-S/nCoV-19 vaccine, the levels of IgM and IgA remained the same as observed after the first dose (Figure 4a, b, d, e, g, h). Only one volunteer showed an IgA level positive between 7 and 30 days (Figure 4e, h).

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Figure 4
Levels of IgM, IgA, and IgG production after the second dose of the ChAdOx1-S/nCoV-19 vaccine. (n= 13) (a-c) Comparison of immunoglobulin production before and 7 days after vaccination. (d-f) Comparison of immunoglobulin production 7 and 15 days after vaccination. (g-i) Comparison of immunoglobulin production 15 and 30 days after vaccination. The round symbols represent women and the square symbols represent men. The dashed line indicates cut-off value. *p < 0.05 represents the difference between 7 and 15 after the second ChAdOx1-S/nCoV-19 dose.

However, IgG levels increased 1.2-fold between day 7 (median 2.044, range 1.210-2.835 OD) and day 15 (median 2.494, range 0.6960-2.735 OD) after the second dose, reaching peak levels (Figure 4f). Between 15 days (median 2.494, range 0.6960-2.735 OD) and 30 days (median 2.225, range 0.9890-2.933 OD), IgG levels remained stable, with no statistically significant difference observed (Figure 4i). Although IgG seroconversion was observed in all volunteers, a progressive 1.2-fold decline in IgG production occurred from 60 days (median 1.283, range 0.9120-2.933 OD) to 120 days (median 1.114, range 0.3250- 1.508 O.D) after the second dose. By this later time point, some individuals exhibited values below the cut-off, as shown in the supplementary material.

The production of IgM, IgA, and IgG (anti-S SARS-CoV-2) antibodies was monitored up to 120 days after the second dose of the ChAdOx1-S/nCoV-19 vaccine (Figures S2a, b, c). No increase in IgM antibody levels was observed after 30, 60, 90, and 120 days following the second dose, as the values were below the cut-off value (0.347; Figure S2a). In general, after 30 days of the second dose of the ChAdOx1-S/nCoV-19 vaccine, there was a decline in IgA levels in most volunteers. Only one participant showed an increase in IgA at 30 days (Figure S2b), followed by a slight decline at 120 days after vaccination. IgG levels peaked 15 days after the second dose of the vaccine, with a median of 2.494 (minimum of 0.696 and maximum of 2.735). Statistical significance was maintained until 90 days after the second dose, at which point the values were no longer significantly high compared to the period before the first dose of the vaccine. At this point, the median was 1.359, with a minimum value of 0.702 and a maximum of 1.637.

Finally, a progressive decline in IgG levels was observed from 60 days after the second dose, with a median of 1.114 (range 0.3250-1.508) (Figure S2c). However, one volunteer exhibited a pronounced decline in IgG production at 120 days after vaccination, falling below the cut-off value (0.687); the group median at this time point was 0.8 (range 0.325-2.1 OD; Figure S2c).

Liver injury is a common feature of COVID-19, and instances of liver injury have been reported following SARS-CoV-2 vaccination. Therefore, to evaluate hepatic health, plasma levels of AST and ALT were analyzed in volunteers after the first and second doses of ChAdOx1-S/nCoV-19. Figure 5a shows a decrease in AST levels 7 days after the first dose (from median 40.2, range 3.6-66.4 U/L to median 4.9, range 1.04-24.4 U/L). No significant differences in AST levels were observed at other intervals, with values remaining low until 30 days after vaccination (Figures 5b, c). ALT levels showed a significant reduction between days 7 and 15 (from median 43.3, range 5.8-57.6 U/L to median 10.2, range 2.9-41.3 U/L) after the first dose (Figure 5e). No differences were identified for ALT at the other time points analyzed (Figures 5d, f).

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Figure 5
Levels of liver enzyme aspartate aminotransferase (AST) and alanine aminotransferase (ALT) before and after first ChAdOx1-S/nCoV-19 vaccination. (n= 13). (a-c) AST levels, and (d-f) ALT levels before 30 days after the first dose of the vaccine. Round symbols represent women and square symbols represent men. The dashed line indicates cut-off value. ***p < 0.001 represents the difference between 0 and 7 days after the first ChAdOx1-S/nCoV-19 dose. *p < 0.05 represents the difference between 7 and 15 days after the first ChAdOx1-S/nCoV-19 doses.

The second dose of ChAdOx1-S/nCoV-19 did not affect until 30 days after vaccination (Figures 6a, b, c). However, ALT levels changed between 15 days (median 4.5, range 1.3-106.4 U/L) and 30 days (median 9.4, range 0.973-42.5 U/L) after vaccination, mainly because of the ALT levels of two volunteers (Figures 6d, e, f).

The AST/ALT ratio is used for the complementary investigation of the etiology underlying liver disease. Therefore, plasma AST/ALT ratios were analyzed before and after the administration of the first and second doses of ChAdOx1-S/nCoV-19. The data revealed that the ratio was 1.2-fold higher at 7 days compared to baseline (day 0; median 2.25, range 0.5-22.3) and declined significantly by 3.2-fold 30 days after the first dose (Figure 7a). Following the second dose, the ratio was 6.1-fold higher 15 days post-vaccination (median 2.450, range 0.1000-7.000; Figure 7b).

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Figure 6
Levels of liver enzyme aspartate aminotransferase (AST) and alanine aminotransferase (ALT) before and after first ChAdOx1-S/nCoV-19 vaccination. (n= 13). (a-c) AST levels, and (d-f) ALT levels before 30 days after the second dose of the vaccine. Round symbols represent women and square symbols represent men. The dashed line indicates cut-off value. *p < 0.05 represents the difference between 15 and 30 days after the second ChAdOx1-S/nCoV-19 dose.
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Figure 7
Evaluation of Ratio of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) before and after ChAdOx1-S/nCoV-19 vaccination. (n= 13) (a) The AST/ALT ratio before to 30 days after the first dose. (b) The AST/ALT ratio before to 30 days after the second dose. Round symbols represent women and square symbols represent men. The dashed line indicates cut-off value. The Kruskal-Wallis tests were used to compare multiple groups of AST/ALT ratio. *p < 0.05.

The levels of direct bilirubin (Figures 8a, d, g and 9a, d, g), indirect bilirubin (Figures 8b, e, h and 9b, e, h), and total bilirubin (Figures 8c, f, i and 9c, f, i) in the plasma at time intervals from 0 to 30 days after the administration of the first and second doses. No statistically significant differences were observed for direct or total bilirubin from day 0 to 30 days the first dose. However, a statistically significant 43-fold increase in indirect bilirubin was observed 7 days after the first dose (median 1.8, range 0.169-15.3 mg/dL), with the exception of two volunteers who showed a reduction in concentration during the same period (Figure 8b). No statistically significant differences were observed at the other time points or after the administration of the second dose (Figure 9).

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Figure 8
Levels of direct, indirect, and total bilirubin before and 30 days after the first dose of the ChAdOx1-S/nCoV-19 vaccine. (n= 13) (a-c) bilirubin levels before and 7 days, (d-f) bilirubin levels 7 and 15 days, and (g-i) bilirubin levels 15 and 30 days. Round symbols represent women and square symbols represent men. The dashed line indicates cut-off value. *p < 0.01 represents the difference between 0 and 7 days after the first ChAdOx1-S/nCoV-19 dose.
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Figure 9
Profile of direct, indirect, and total liver enzyme levels among the 13 participants evaluated before and 30 days after the second dose of the ChAdOx1-S/nCoV-19 vaccine. Graphs (a-c) show bilirubin levels before and 7 days after vaccination, (d-f) bilirubin levels at 7 and 15 days, and (g-i) bilirubin levels at 15 and 30 days. Circles represent female participants and squares represent male participants. The cutoff value is indicated by the dashed line. Statistical significance was calculated using the Mann-Whitney test.

IL-6 is a major cytokine produced in response to SARS-CoV-2 infection, and elevated levels have been correlated with liver injury. To verify the IL-6 levels, plasma from volunteers was analyzed before and after the first and second doses ChAdOx1-S/nCoV-19. Figure 10 shows the plasma levels of the pro-inflammatory cytokine IL-6 after vaccination. Although no statistically significant differences were observed between the periods analyzed, median values decreased 15 days after the first dose (median 54, range 0-805 pg/mL) and increased after the second dose (median 80, range 22-592 pg/mL; Figures 10a, b).

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Figure 10
Plasma levels of IL-6 in 13 participants after vaccination. (a) Cytokine measurement 7 to 15 days after the first dose. (b) Cytokine measurement 7 to 15 days after the second dose. Round symbols represent women and square symbols represent men. The dashed line indicates the cut-off value.

Principal Component Analysis (PCA) was performed to analyze the overall pattern and similarity among immunological and biochemical responses following vaccination. PC1 explained 28.1% of the total variance, while PC2 accounted for 19.2%, together representing 47.3% of the cumulative variance. Supplementary Figure S3 presents the PCA of the biochemical and immunological parameters examined in this study. The analysis did not reveal a clear separation between samples collected after the first and second vaccine doses; however, a gradual shift across the PCA space was observed. Among the analyzed variables, IgG, IgA, and time post-vaccination contributed most strongly to the variance, showing a predominant influence along PC1 and clustering in the lower right quadrant of the PCA space (Figure S3). Samples collected after the first dose were more widely dispersed, whereas samples collected after the second dose appeared more concentrated. The AST/ALT ratio contributed in an opposing direction relative to the humoral response variables. Although IgA levels did not exceed the cut-off, their contribution to the PCA indicates variability among individual samples.

DISCUSSION

In this study, we aimed to evaluate the serological response and adverse reaction symptomatology of volunteers before and after receiving the ChAdOx1-S/nCoV-19 vaccine, with particular focus on seroconversion and potential liver alterations across different time points of blood collection. Most volunteers experienced adverse reactions, typically beginning on the first day, with local (injection site pain) and systemic symptoms (fever, myalgia, and headache) being the most frequent. These findings are consistent with large clinical trials (Voysey et al. 2021b, Folegatti et al. 2020), which also identified these events as the most common. Moreover, in agreement with Gopaul et al. (2022), our data revealed a reduction in the intensity and frequency of adverse events after the second dose compared to the first, suggesting that the ChAdOx1-S/nCoV-19 vaccine not only enhances the immune response but also improves tolerability, an important factor for vaccine safety, compliance, and uptake.

Among the adverse reactions, injection site pain, fever, myalgia, and headache were the most commonly reported, consistent with the findings of Folegatti et al. (2020), who also highlighted these as the most common. Regarding the intensity and frequency of adverse events, Gopaul et al. (2022) reported a significant reduction in the second dose compared to the first dose as observed by our study. These observations suggest that the second dose of ChAdOx1-S/nCoV-19 vaccine not only enhances the immune response but also reduces the incidence of adverse events, potentially promoting higher vaccine compliance. This reduction in adverse events after the second dose is an important consideration when discussing vaccine safety and efficacy, as it may support public confidence and facilitate greater acceptance of vaccination programs.

Regarding immunogenicity, the antibody response to ChAdOx1-S/nCoV-19 was predominantly mediated by IgG, with minimal contributions from IgM and IgA. By 15 days post-vaccination, IgG levels had increased significantly, emphasizing the key role of IgG in viral neutralization and long-term immunity. Although an IgA activation was detected, levels remained below the cut-off, indicating a limited mucosal contribution. The second dose contributed to increased IgG levels, with peak values observed between 7 and 15 days, reinforcing the vaccine’s ability to induce a strong adaptive immune response. These findings are consistent with those of Pozzetto et al. (2021), who also reported higher IgG titers after the second dose, highlighting the importance of completing the vaccination schedule for optimal protection. Despite the high IgG response, antibody levels began to decline after 60 days, with some individuals reverting to seronegativity at 120 days. This pattern of an initial increase followed by a gradual decline has been previously reported; however, this waning does not necessarily imply a complete loss of immune protection. Cellular responses mediated by T lymphocytes play a critical role in clearing virus-infected cells, establishing long-term immune memory, and responding to viral mutations (Chen et al. 2025). These findings underscore the need for continued immune surveillance and suggest the need of booster doses, particularly given the emergence of new SARS-CoV-2 variants.

Additionally, vaccine-induced immune activation can trigger systemic inflammatory responses that may impact liver function. We observed a significant increase in the AST/ALT ratio 7 days after the first dose, suggesting an inflammatory response or vaccine-related hepatic effect. This finding aligns with previous reports of liver injury patterns during both SARS-CoV-2 infection and post-vaccination, in which elevations in AST, ALT, and bilirubin have been documented (Nardo et al. 2021, Shroff et al. 2022). However, the AST/ALT ratio returned to baseline levels by day 15 after the first dose and by day 30 after the second dose, indicating that these alterations were transient.

In parallel, a statistically significant and clinical increase in indirect bilirubin was detected 7 days after the first dose, whereas no significant fluctuations were observed after the second dose. This suggests that bilirubin metabolism is affected only during the initial immune activation, with stabilization thereafter. Individual variability was also noted, as two volunteers exhibited a reduction in bilirubin levels over the same period. These findings emphasize the importance of monitoring liver function during vaccination process, particularly in individuals with pre-existing liver conditions.

Considering the role of inflammation in these changes, we also analyzed IL-6, a cytokine central to both immune regulation and hepatocyte function (Hunter & Jones 2015). Although IL-6 levels did not vary significantly across time points, we observed a decrease in median values 15 days after the first dose, which may reflect regulatory control following initial activation. Conversely, IL-6 levels increased after the second dose, possibly indicating a stronger immune response aimed at consolidating memory and antibody production rather than causing liver injury. This dual role of IL-6, as both a pro-inflammatory and resolution mediator, has been highlighted in previous studies (Tanaka et al. 2014), supporting its interpretation as part of the physiological adaptation to vaccination rather than a marker of hepatic damage.

Therefore, the interpretation of IL-6 variations requires a comprehensive analysis within the broader immunological and clinical context. The lack of significant differences in IL-6 levels suggests that ChAdOx1-S/nCoV-19 vaccination does not induce an exacerbated inflammatory response, which is an important finding given concerns about potential adverse reactions. Our data suggest that ChAdOx1-S/nCoV-19 vaccination induces IL-6 fluctuations consistent with immune activation without clear evidence of persistent liver injury.

Multivariate analysis further supported these findings. While PCA did not reveal a clear distinction between samples collected after the first and second vaccine doses, a gradual shift in multivariate space was observed. This indicates progressive changes in the combined immunological and biochemical responses following vaccination. This pattern likely reflects the temporal dynamics of the immune response, particularly those associated with antibody production, which substantially contributed to the overall dataset variability. Notably, the wider dispersion observed after the first dose indicates greater inter-individual variability in early responses, whereas the more clustered pattern after the second dose may reflect a more coordinated adaptive immune response. This is consistent with previous studies reporting heterogeneous antibody responses after a single dose, as well as increased population homogeneity following booster vaccination (Faro-Viana et al. 2022). The strong contribution of IgG, IgA, and time post-vaccination emphasizes the pivotal role of humoral response development over time. Additionally, the inverse relationship between the AST/ALT ratio and antibody-related variables is consistent with the transient systemic effects observed shortly after vaccination. However, as the first two principal components explained less than half of the total variance, additional sources of variability, including clinical heterogeneity and individual factors, likely contributed to the overall dispersion.

In conclusion, this study demonstrates that the ChAdOx1-S/nCoV-19 vaccine was well-tolerated and elicited a high IgG response following the first and second doses, which declined by 120 days. Furthermore, the vaccine showed minimal hepatic impact in the evaluated population. However, the study was limitated by a relatively high dropout rate attributed to SARS-CoV-2 infection and the intensive blood sampling schedule.

SUPPLEMENTARY MATERIAL

Figures S1-S3.

Acknowledgements

The authors are glad to Marcela Espindola Palmeira Pereira, Raphael de Melo Carpes, Artur Nunes Paes and David Alves Vieira of the Institute of Biodiversidade and Sustentabilidade (NUPEM/UFRJ) which contribute to the study; This study was funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Grant N°: 260003/012799/2021, N°: 26/210822/2021.

  • Data availability
    The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.

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Edited by

  • Handling editor
    João Duarte

Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Publication Dates

  • Publication in this collection
    28 Aug 2026
  • Date of issue
    2026

History

  • Received
    24 Oct 2025
  • Accepted
    31 Mar 2026
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