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Heliyon
Heliyon
Heliyon
2405-8440
Elsevier

S2405-8440(24)12641-2
10.1016/j.heliyon.2024.e36610
e36610
Research Article
Cellular immune response to a single dose of live attenuated hepatitis a virus vaccine in obese children and adolescents
Soponkanabhorn Tanatchabhorn a1
Suratannon Narissara b1
Buranapraditkun Supranee cd
Tubjareon Chomchanat a
Prachuapthunyachart Sittichoke a
Eiamkulbutr Sutha e
Chongsrisawat Voranush voranush.c@chula.ac.th
a⁎
a Department of Pediatrics, Faculty of Medicine, Chulalongkorn University and King Chulalongkorn Memorial Hospital, The Thai Red Cross Society, Bangkok, Thailand
b Center of Excellence for Allergy and Clinical Immunology, Division of Allergy, Immunology and Rheumatology, Department of Pediatrics, Faculty of Medicine, Chulalongkorn University and King Chulalongkorn Memorial Hospital, The Thai Red Cross Society, Bangkok, Thailand
c Division of Allergy and Clinical Immunology, Department of Medicine, Faculty of Medicine, Chulalongkorn University and King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok, Thailand
d Center of Excellence in Vaccine Research and Development (Chula Vaccine Research Center-Chula VRC), Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
e Excellence Center of Organ Transplantation, King Chulalongkorn Memorial Hospital, The Thai Red Cross Society, Bangkok, Thailand
⁎ Corresponding author. voranush.c@chula.ac.th
1 Tanatchabhorn Soponkanabhorn and Narissara Suratannon are co-first authors.

20 8 2024
30 8 2024
20 8 2024
10 16 e3661023 8 2023
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© 2024 The Authors. Published by Elsevier Ltd.
2024

https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
Background

Limited data are currently available regarding the cellular immune response to a live attenuated hepatitis A virus (HAV) vaccine, especially in children with obesity. The objective of this retrospective cohort study was to compare the activation of antigen-specific interferon (IFN)-γ+ T cells in obese children and adolescents with healthy individuals before and after immunization with a single dose of live attenuated HAV vaccine.

Methods

Blood samples were obtained from the 2021 study by Dumrisilp et al. investigating the immunogenicity of the live attenuated hepatitis A vaccine in children and young adults. Prior to enrollment, all 212 subjects had never received any HAV vaccine and tested negative for anti-HAV antibodies. The participants were vaccinated with a freeze-dried, live attenuated HAV vaccine of the H2 strain. In this study, we analyzed the stored peripheral blood mononuclear cells (PBMCs) obtained from a subgroup of 30 obese subjects and 30 normal-weight healthy controls of the same age and sex. PBMCs were collected before and 8–9 weeks after HAV vaccination for further analysis. These cells were stimulated with a recombinant antigen derived from HAV-VP3, and the immune response was evaluated using the IFN-γ enzyme-linked immunospot (ELISpot) assay.

Results

The between-group analysis indicated that the T-cell response of obese participants was comparable to that of normal-weight controls both before and after vaccination. The change in IFN-γ production from before to after vaccination in the obese group was not significantly different from that of the control group. Additionally, in the obese group, no correlation was found between IFN-γ production and clinical characteristics such as sex, body mass index, waist circumference, and acanthosis nigricans.

Conclusion

Testing for cellular immune response provides a comprehensive understanding of the overall immune response to vaccination. This study, the first to explore this significant aspect, suggests that obesity does not affect the short-term cellular immune response to live attenuated HAV vaccination.

Keywords

Obesity
Live attenuated hepatitis a virus vaccine
T cells
Cellular immunity
Children
Adolescents
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pmc1 Introduction

Obese individuals are at an increased risk of developing nonalcoholic fatty liver disease (NAFLD) compared to the normal-weight population. A meta-analysis revealed that the prevalence of NAFLD in obese children and adolescents was much higher than their normal-weight counterparts: 36.1 % and 2.3 %, respectively [1]. NAFLD can progress to severe chronic liver disease (CLD), such as cirrhosis and hepatocellular carcinoma, which increases susceptibility to severe hepatitis A virus (HAV) infection. Therefore, obese people including children and adolescents should receive the vaccine to prevent severe HAV hepatitis [2].

Currently, the HAV vaccine is available in two forms: inactivated and live-attenuated. The efficacy of both vaccine types has been well recognized [3]. The live attenuated HAV vaccine has been recommended as part of the Chinese Expanded Immunization Program since 2008 to prevent the spread of HAV [4]. Additionally, the Indian Academy of Pediatrics has recommended using a single dose of the H2-strain live attenuated HAV vaccine instead of double doses since 2014 [5].

It has been reported that obese individuals develop a poorer immune response to vaccination compared to the general population [[6], [7], [8], [9], [10]]. A study investigating the immune response to the inactivated trivalent influenza vaccine (TIV) in adults found that higher body mass index (BMI) was initially correlated with a greater antibody response but was associated with an accelerated waning of antibody levels at 12 months post-vaccination [11]. In addition, obese individuals have been shown to have lower levels of specific CD8+ T cells and reduced interferon (IFN)-γ production [11]. As a result, they may be at increased risk for vaccine-preventable diseases, as well as a higher risk of developing severe infections and death from certain respiratory illnesses, including H1N1 influenza [12] and COVID-19 [13,14]. Childhood obesity has also been linked to an increased risk of severe COVID-19 [15]. The 2022 study by Tubjaroen et al. on the immunogenicity of an mRNA-based COVID-19 vaccine found that obese adolescents had significantly lower levels of anti-SARS-CoV-2 spike protein antibodies after receiving two doses of the vaccine compared to healthy controls [16]. However, there are conflicting data on the immunogenicity of the HAV vaccine in obese individuals. Regarding humoral immune response, studies in adults have shown that higher BMI is inversely correlated with antibody responses to the inactivated HAV vaccine [17,18]. A study in older individuals (mean age 55.2 years) who received a combined inactivated HAV/HBV vaccine showed that high BMI was the most significant factor associated with a substantial waning of HAV antibody [18]. Another study in adults found that elevated body weight and BMI were significant predictors of having non-protective levels of anti-HAV IgG four weeks after receiving the inactivated HAV vaccine [17]. In contrast, a study showed no difference in the immunogenicity of the inactivated HAV vaccine between healthy and obese young adults in Korea [19]. This finding is consistent with the 2021 study by Dumrisilp et al., which found comparable humoral immune responses to the live attenuated HAV vaccine between obese children/young adults and normal-weight participants [20].

Regarding cellular immunity, previous studies have highlighted the critical role of virus-specific T lymphocytes in combating hepatitis A infections and limiting liver injury [21]. Current data indicate that impairments in vaccine-induced cellular immune responses among obese individuals include decreased natural killer cell cytotoxicity, serum cytokine and IFN-γ levels, as well as diminished CD8+ and CD4+ T cell functionality in long-term immune responses [11,[22], [23], [24], [25]]. For instance, Costanzo et al. observed reduced peripheral blood γδ T cells in obese subjects, which are pivotal in limiting viral replication through IFN-γ production [26]. However, research on cellular responses in obese individuals has primarily relied on evidence from natural infections or vaccination with inactivated vaccines.

Inactivated vaccines, which consist of killed viruses, interact differently with the immune system and typically induce weaker cellular immunity. Live attenuated vaccines, resembling live viruses, have the potential to prime a stronger cellular immune response. Currently, the impact of obesity on the cell-mediated immune response to the live attenuated HAV vaccine, particularly in children, remains poorly understood.

This significant knowledge gap motivated our study, which aimed to compare the cellular immune response following a single dose of the live attenuated HAV vaccine between obese and healthy children/adolescents. Additionally, we sought to explore the association between clinical characteristics and post-vaccination cellular immune responses in obese participants.

2 Methods

2.1 Study population and samples

This retrospective cohort study obtained blood samples from the 2021 study by Dumrisilp et al. titled “Impact of Obesity and Being Overweight on Immunogenicity to Live Attenuated Hepatitis A Vaccine in Children and Young Adults” [20]. The study recruited a total of 212 participants, ranging in age from 7 to 25 years. Participants were recruited from two primary schools, one secondary school, one university in Bangkok, and the outpatient obesity clinic of King Chulalongkorn Memorial Hospital between November 2019 and February 2020. Prior to enrollment, all subjects had never received any HAV vaccine and were seronegative for anti-HAV (anti-HAV <20 mIU/mL). Participants were all vaccinated with the H2 strain, freeze dried, live attenuated HAV vaccine (MEVAC™-A). Peripheral blood mononuclear cells (PBMCs) were collected before and 8–9 weeks after HAV vaccination for subsequent analysis.

For this study conducted between November 2021 and February 2022, we analyzed stored PBMCs collected from 30 obese subjects aged 8–14 years (15 males) and 30 normal-weight healthy controls of the same age and sex. The number of study participants was determined by feasibility of immunological analyses and enrollment. The disposition of subjects is illustrated in Fig. 1. Obesity and normal weight were classified based on body mass index (BMI) using the WHO Child Growth Standards [27]. The waist circumference Z-score was calculated based on the international percentile cutoffs for waist circumference in children and adolescents with normal weight [28]. This study was registered in the Thai Clinical Trials Registry (TCTR) (ID number 20210825005).Fig. 1 Disposition of subjects in the study.

Fig. 1

2.2 Peripheral blood mononuclear cells (PBMCs) isolation

Peripheral blood mononuclear cells (PBMCs) were isolated from the acid citrate dextrose (ACD) blood of participants using density gradient separation. First, peripheral blood was diluted and mixed with RPMI1640 medium (Gibco, Germany) at a ratio of 1:1. Next, the diluted blood was gently layered on top of 4 mL of Isoprep (Robbins Scientific Corporation, Sunnyvate, CA). After centrifugation at 2200 rpm for 20 min at room temperature, PBMCs were collected and transferred to a new tube. We then washed the PBMCs twice with RPMI1640 medium, and after counting the cells, we cryopreserved them with freezing media containing 10 % dimethyl sulfoxide (DMSO; Sigma, USA) and 90 % fetal bovine serum (FBS; Bio Whittaker, Maryland, USA). Finally, the frozen PBMCs were transported to a liquid nitrogen tank for further study.

2.3 Thawing the PBMCs

The cryopreserved PBMCs were thawed in a 37 °C water bath, and then transferred into a 15 mL conical centrifuge tube. Next, we added cold R10 medium (RPMI1640 supplemented with 100 U/mL of penicillin, 100 U/mL of streptomycin, and 10 % FBS). After washing, we counted the cells and adjusted them to a concentration of 5 × 106 cells/mL with R10 medium for ELISpot assay. The viability of PBMCs was confirmed to be more than 80 % post-thaw recovery, determined using trypan blue dye exclusion. This ensured that a sufficient number of viable cells were available for analysis.

2.4 Assessment of cellular immune response

We stimulated PBMCs of both groups before vaccination and at 8–9 weeks after vaccination with the recombinant antigen of HAV-VP3 (MyBioSource, San Diego, CA, USA) at concentrations of 5 and 10 μg/mL, respectively. Since the dose of recombinant HAV-VP3 that can successfully stimulate the IFN-γ response has not been studied previously, we performed preliminary experiments to find the optimal concentration, including 2 μg/mL, 5 μg/mL, and 10 μg/mL (data not shown). The suitable concentrations to stimulate the IFN-γ response are 5 and 10 μg/mL, respectively. We then quantified T cell responses using the human IFN-γ Enzyme-Linked Immunospot (ELISpot) assay.

Briefly, 96-well nitrocellulose membrane plates (MAIP 4510, Millipore, USA) were coated overnight with monoclonal antibody anti-human IFN-γ (1-D1K, Mabtech, Stockholm, Sweden). The plates were then washed with 1 × PBS and blocked with complete medium (RPMI-10 % FBS). A total of 250,000 PBMCs were added to each well and cultured with the recombinant VP3 antigen of the HAV at 37 °C with 5 % CO2 for 40 h. The complete medium alone and phytohemagglutinin (PHA) served as a negative and positive control well, respectively. After incubation, 1 μg/mL anti-human IFN-γ biotinylated (7-B6-1 biotinylated, Mabtech, Stockholm, Sweden) monoclonal antibody was added and incubated for 3 h at room temperature (RT). After washing, a 1:1000 dilution of streptavidin-alkaline phosphatase (ALP, Mabtech, Stockholm, Sweden) was added to each well and incubated for 1 h at RT. The wells were then washed and developed by adding 100 μL of substrate solution (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium, BCIP/NBT) to each well and incubated for 10–20 min at RT/dark or developed until distinct spots emerged. The reaction was stopped by washing the plate in tap water and leaving the plate to dry. The spots were counted using an immunospot analyzer (S6 Ultimate, CTL, Germany). HAV-VP3-specific PBMCs were calculated by subtraction of background and expressed as spot-forming units (SFUs) per 106 PBMCs.

2.5 Statistical analysis

Statistical analysis was performed using STATA 15.1 software (StataCorp, College Station, Tx, USA). Categorical data were presented as numbers and percentages. Continuous data were reported as mean with standard deviation (SD) and median with interquartile range (IQR). Comparison between the two groups was analyzed using the two-sample independent t-test for geometric means and Wilcoxon rank sum test for medians. The correlation between baseline characteristics and post-vaccination IFN-γ production in the obese group was analyzed using quantile regression. P-values less than 0.05 were considered statistically significant.

2.6 Ethical considerations

The study protocol was approved by the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (Approval Certificate number 923/63, Date of Approval March 5, 2021). The study was conducted in accordance with the ICMR guidelines for biomedical research on human subjects and the 1964 Declaration of Helsinki and its later amendments. Written informed consent and assent for research on stored biological samples were obtained from participants and/or their parents concurrently with the 2021 study by Dumrisilp et al. [20]. The samples were de-identified and anonymous.

3 Results

3.1 Baseline characteristics of participants

The mean age of 60 participants was 11 ± 1.5 years, with 15 males in each group. Body weight, BMI Z-score, and waist circumference of both groups are shown in Table 1. In the obese group, 21 out of 30 participants (70 %) had acanthosis nigricans, and all obese children had truncal obesity.Table 1 Participant characteristics by nutritional status groups.

Table 1	Control group	Obese group	
(n = 30)	(n = 30)	
Weight (kilogram)	33.5 ± 6.5	65.5 ± 14.5	
BMI Z-score	−0.3 (0.6)	3.1 (1)	
Waist circumference (cm)	58.1 ± 12.2	89.4 ± 9.5	
Presence of acanthosis nigricans	0	21 (70)	
Presence of truncal obesity	0	30 (100)	
Postvaccination anti-HAV titers (mIU/mL)	374.5	498.1*	
(334.4–419.3)	(406–610.9)	
Data expressed as mean ± SD, number (percentage), or geometric mean titer (95 % confidence interval).

*P = 0.02.

3.1.1 Cellular immunogenicity before and after vaccination

Overall, the HAV-VP3 antigen at concentrations of 5 and 10 μ g/mL was able to induce IFN-γ production of T cells in both groups as shown in Table 2. Although females had a higher IFN-γ production than males, the difference was not statistically significant (data not shown).Table 2 Comparison of interferon-γ production of T cells after stimulation with recombinant VP3 antigen of the hepatitis A virus in control and obese groups.

Table 2HAV-VP3 antigen concentration	Interferon-γ production (SFUs per 106 PBMCs)	
Control group (n = 30)	Obese group (n = 30)	
Prevaccination	Postvaccination	P-value	Prevaccination	Postvaccination	P-value	
5 μ g/mL	148 (72–228)	160 (36–348)	0.43	88 (8–216)	140 (68–380)	0.08	
10 μ g/mL	120 (72–276)	230 (36–380)	0.19	126 (12–216)	184 (108–316)	0.04	
Data expressed as median (IQR).

The T cell response after vaccination was significantly higher than before vaccination only in the obese group, specifically with HAV-VP3 stimulation at a concentration of 10 μ g/mL (P = 0.04).

The between-group analysis revealed that the T cell response of obese participants was comparable to that of normal controls both before and after vaccination (Fig. 2A, B, 2C). The change in IFN-γ production from before to after vaccination in the obese group was not significantly different from that of the control group (Table 3).Fig. 2 Comparison of interferon-γ production between controls and obese subjects after stimulation with recombinant VP3 antigen of hepatitis A virus at concentration of (A) 5 μg/mL and (B) 10 μg/mL, (C) Representative panel of T cells by interferon-γ ELISpot assay in control and obese groups.

Fig. 2

Table 3 The change in IFN-γ production from before to after vaccination.

Table 3HAV-VP3 antigen concentration	Change in interferon-γ production from before to after vaccination (SFUs per 106 PBMCs)	P-value	
Control group	Obese group	
(n = 30)	(n = 30)	
5 μ g/mL	18 (−100 to 144)	42 (−12 to 128)	0.54	
10 μ g/mL	32 (−60 to 136)	66 (−44 to 128)	0.63	
Data expressed as median (IQR).

In the obese group, there was no correlation between IFN-γ production and clinical characteristics such as sex, BMI Z-score, waist circumference Z-score, and acanthosis nigricans (Table 4).Table 4 Correlation between clinical characteristics and postvaccination IFN-γ production in the obese group.

Table 4	Coefficient (95 % CI)	R2	P-value	
Male	−60 (−272.9, 152.9)	0.01	0.57	
Body mass index Z-score	−57.8 (−147.2, 31.6)	0.05	0.20	
Waist circumference Z-score	−34 (−92.4, 24.4)	0.05	0.24	
Acanthosis nigricans	−180 (−424.8, 64.8)	0.08	0.14	

4 Discussion

The prevalence of HAV infection is continuously declining in many parts of the world due to factors such as higher socioeconomic status, improved sanitary conditions, and widespread availability of HAV vaccines. Typically, symptomatic HAV infection occurs in older children and adults. Consequently, outbreaks of HAV infection can occur in areas with lower rates of HAV infection and low HAV vaccine coverage [7]. While most individuals infected with HAV recover completely and acquire lifelong immunity, there is a small proportion, especially those with chronic liver disease, who are at risk of developing fulminant hepatitis. Therefore, it is crucial for individuals in this group to receive HAV vaccination [6].

An increasing body of evidence supports the excellent immunogenicity of both inactivated and live attenuated HAV vaccines. Notably, the live attenuated vaccine not only induces short-term humoral immunity but also provides long-term protection [8,9,20,[29], [30], [31], [32]]. A study involving 31 pediatric participants examined the immune response 17 years after a single dose of live attenuated HAV vaccination. The findings revealed an anamnestic response in both B cells and T cells at two weeks after receiving a booster dose [29]. Furthermore, the response of memory T cells to HAV antigen stimulation was comparable between subjects with detectable and undetectable anti-HAV antibodies before receiving a booster dose. In contrast, a study involving 52 adults who had undetectable anti-HAV antibodies 10 years after a complete primary inactivated HAV vaccination revealed that the response of memory T cells correlated with the level of antibody production after the booster dose [33]. The varying association between memory T cell and B cell responses may be attributed to age differences, resulting in diverse immune responses. The mean age of participants in the former study was approximately 21 years, whereas in the latter study, it was 61 years. Consequently, in the former study, 11 out of 13 participants (84.6 %) who had undetectable antibodies before the booster dose became seroconverted, whereas in the latter study, 40 out of 52 participants (76.9 %) had detectable HAV antibodies after the booster dose.

Testing for cellular immune response offers a comprehensive understanding of the overall immune response to vaccination, especially for intracellular pathogens such as viruses. Assessing vaccine-induced cellular memory response is crucial for predicting the duration of protection and determining the need for a booster dose. This information is particularly important for vaccine design targeting medically vulnerable groups, including individuals who are obese. However, there is limited data available on the cellular immune response to a live attenuated HAV vaccine, especially in children with obesity.

In this study, T lymphocytes were stimulated using a recombinant HAV-VP3 antigen, which contains 245 amino acids derived from the HAV-VP3 capsid protein sequence. This antigen was chosen for its strong immunogenic properties [34,35]. Two different concentrations of the HAV-VP3 antigen were utilized to analyze T cell stimulation, revealing that obese subjects exhibited a comparable magnitude of T cell response to that of healthy controls, even with a low-dose antigen. The measurement of T cell numbers was performed using the IFN-γ ELISpot assay, which serves as a surrogate for assessing cellular immune response. The ELISpot assay is a cost-effective, highly sensitive, and easy-to-perform method. The IFN-γ ELISpot assay requires a lower number of T cells compared to the intracellular cytokine staining (ICS) assay, which can identify the phenotype of responding T cells. In a recent study examining the T cell response to SARS-CoV-2 after COVID-19 infection, a good correlation was observed between the ELISpot and ICS assays [36]. Notably, the ELISpot assay exhibited superior sensitivity in detecting T cell responses. The authors suggested that the ELISpot assay may be suitable for analyzing immunogenicity in vaccine trials. However, there is evidence showing a lack of correlation between IFN-γ production and BCG-induced immune protection against tuberculosis [37]. Therefore, further investigation is needed to elucidate the role of antigen-specific cytokine-secreting T cells as a protective immune marker in HAV vaccination.

The present study represents the first exploration of cellular immunity in obese children and adolescents against HAV vaccine. In the 2021 study by Dumrisilp et al., all obese subjects and normal controls achieved seroprotection after receiving a single dose of live attenuated HAV vaccine [20]. Thus, we aimed to investigate the cellular immune responses of the subjects at the same time point as we measured HAV serology in the previous study. This was done to understand the cellular immune response as reflected in IFN-γ levels against HAV. Our results revealed that the live attenuated HAV vaccine can elicit a comparable short-term cellular immune response in obese individuals to that observed in healthy subjects. This effect is potentially attributable to the robust cellular immune response induced by the live attenuated HAV vaccine, which mimics natural infections [38]. The detection of IFN-γ-producing cells in pre-vaccinated PBMCs (shown in Table 2 and Fig. 2) might result from IFN-γ from possible exposures to other viruses (e.g. polioviruses/rotaviruses containing similar amino acid sequences as VP3 antigen of HAV [39,40]. Nonspecific IFN-γ can also be produced by innate immune cells such as natural killer cells or IFN-γ-producing type 1 innate lymphoid cells [41]. These reasons also emphasized the strong need of the background IFN-γ at pre-vaccinated stage, ensuring that the post-vaccinated IFN-γ levels found are specific to HAV.

Metabolic syndrome, which is closely linked to overweight and obesity, is characterized by the presence of at least three out of five cardiometabolic parameters. These parameters include abdominal obesity, hypertension, hyperglycemia/insulin resistance, high serum triglycerides, and low serum high-density lipoprotein [42]. A study demonstrated that young adults with obesity and metabolic syndrome exhibited various alterations in immune cell populations, such as increased total lymphocytes and decreased granulocytes and NK lymphocytes [43]. Moreover, increasing evidence indicates that metabolic syndrome contributes to reduced vaccine effectiveness [6]. A recent study investigated the impact of cardiometabolic risk factors on serum cytokines and chemokines following influenza vaccination in overweight and obese children [44]. The children were categorized into two groups: metabolically healthy and metabolically unhealthy, based on the presence of at least two out of five cardiometabolic risk factors. Interestingly, both groups of overweight and obese children, as well as normal-weight children, exhibited similar influenza antibody responses, suggesting that the efficacy of vaccination is not affected by obesity or metabolic health status. However, the metabolically unhealthy individuals showed lower levels of IL-13, a cytokine secreted by various immune cells, compared to the metabolically healthy and normal-weight participants. This finding suggests that metabolic impairment may be associated with an altered T-cell response.

A limitation of this study is the absence of data regarding cardiometabolic risk factors specifically in obese participants. However, our results demonstrated no correlation between post-vaccination IFN-γ production and measures such as waist circumference or acanthosis nigricans – a cutaneous marker of insulin resistance [45] – among the obese participants. This finding suggests that metabolic impairment may not have a significant impact on cellular immune response. Further studies investigating the influence of cardiometabolic risk factors on the memory T cell response will provide insights into long-term vaccine immunity in metabolically unhealthy obese individuals. In addition, our study is limited by its small sample size. Further research involving larger populations, incorporating more comprehensive immunological cellular measurements, and extending the follow-up period for immune parameters would provide greater insight into this important issue. Non-alcoholic fatty liver disease (NAFLD), a common comorbidity in obese individuals, is considered the hepatic manifestation of metabolic syndrome [46]. A study investigating the impact of NAFLD on the outcomes of COVID-19 infection revealed a higher risk of progression to severe COVID-19 and longer viral shedding time in patients with NAFLD [47]. Regarding the immune response to the hepatitis B virus (HBV) vaccine among children with NAFLD, there have been few studies with conflicting results. A study involving 54 obese children with NAFLD demonstrated a similar seroprotection rate compared to children without liver diseases [48]. Conversely, another study demonstrated an exceptionally high rate of nonimmunity against HBV in children with NAFLD, regardless of BMI [49]. Currently, there is no available data on the impact of NAFLD on the immune response to the HAV vaccine. Since most obese subjects in this study were recruited from schools and no data regarding the presence of NAFLD were available, further studies are crucial to explore this aspect. Understanding the immune response in this vulnerable group is essential for designing an effective immunization program against HAV.

Another limitation of this study is the relatively long interval between vaccination and the subsequent immunogenicity testing. Previous studies have suggested that the optimal time point for assessing cellular responsiveness following vaccination is typically around 1–2 weeks after booster vaccination [29,33,50]. In our study, the evaluation of cellular immune response utilized stored PBMCs collected 8–9 weeks after vaccination, coinciding with the measurement of humoral immunity. The extended period between vaccination and evaluation may explain the absence of substantial enhancement in vaccine-induced cellular immunity among most participants. Considering the extended interval in our study, it is important to interpret the results with caution. To ensure the availability and functionality of T cells, we also conducted IFN-γ ELISpot assays on PBMCs from all specimens, including negative and PHA-positive controls. Thus, the lack of a significant increase in IFN-γ production was not attributed to PBMC degradation.

5 Conclusion

The results of this study indicate that obesity does not have an impact on the short-term cellular immune response to live attenuated HAV vaccination. In the obese group, no correlation was found between the cellular immune response and clinical characteristics such as waist circumference, truncal obesity, and acanthosis nigricans. However, further research is warranted to investigate the effects of obesity, cardiometabolic risk factors, and NAFLD on the immune response to the HAV vaccine.

Funding

This work was supported by the Ratchadapiseksompotch Fund, 10.13039/501100002873 Chulalongkorn University, Thailand [grant number RCU_H_64_005_30 ].

Data availability statement

The data used to support the findings of this study are available from the corresponding author upon request.

CRediT authorship contribution statement

Tanatchabhorn Soponkanabhorn: Writing – original draft, Visualization, Validation, Resources, Investigation, Formal analysis, Data curation, Conceptualization. Narissara Suratannon: Writing – review & editing, Visualization, Validation, Resources, Investigation, Formal analysis, Data curation, Conceptualization. Supranee Buranapraditkun: Writing – review & editing, Visualization, Validation, Software, Methodology, Investigation, Formal analysis, Data curation. Chomchanat Tubjareon: Writing – review & editing, Visualization, Resources, Formal analysis, Data curation. Sittichoke Prachuapthunyachart: Writing – review & editing, Visualization, Resources, Formal analysis, Data curation. Sutha Eiamkulbutr: Writing – review & editing, Visualization, Resources, Formal analysis, Data curation. Voranush Chongsrisawat: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The authors would like to thank the participants from Rajavinit Mathayom School and Wat Prayurawongsawat School as well as the staff of Pediatric Outpatient Department at King Chulalongkorn Memorial Hospital. We are also grateful to Dr. Tim Spitzenberger, PhD for editing the manuscript.
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