Heterologous immunization targeting the CST1 antigen confers better protection than ROP18 in mice

Abstract

Aim: To evaluate the protective efficacy induced by heterologous immunization with recombinant baculoviruses or virus-like particles targeting the CST1 and ROP18 antigens of Toxoplasma gondii.

Materials & methods: Recombinant baculovirus and virus-like particle vaccines expressing T. gondii CST1 or ROP18 antigens were developed to evaluate protective immunity in mice upon challenge infection with 450 Toxoplasma gondii (ME49).

Results: Immunization with CST1 or ROP18 vaccines induced similar levels of T. gondii-specific IgG and IgA responses. Compared with ROP 18, CST1 vaccine showed better antibody-secreting cell response, germinal center B cell activation, and significantly reduced brain cyst burden and body weight loss.

Conclusion: Our findings suggest that CST1 heterologous immunization elicited better protection than ROP18, providing important insight into improving the toxoplasmosis vaccine design strategy.

Graphical Abstract

Plain language summary

Article highlights.

• The protective efficacies of a heterologous immunization strategy involving recombinant baculovirus (rBV) and virus-like particle (VLP) vaccines expressing either ROP18 or CST1 antigens were evaluated.

• Sequentially administering rBV and VLP vaccines induced Toxoplasma gondii-specific antibody responses in mice.

• Vaccinated mice experienced substantially less inflammation than the unimmunized control mice upon challenge infection with T. gondii (ME49).

• Both ROP18 and CST1 vaccines reduced the overall parasite burden and promoted the well-being of mice.

• Both rBVs and VLPs expressing either of the two antigens can aid T. gondii vaccine development.

1. Introduction

Toxoplasma gondii is a ubiquitous parasitic organism that affects humans and other warm-blooded vertebrates, often resulting in asymptomatic infections in healthy individuals. However, T. gondii infection can elicit complications that can be fatal depending on the immune status of the affected individual and their age, which includes visual impairment, brain abnormality, impaired fetal development and miscarriage [1]. T. gondii is mainly transmitted through ingestion of cyst-contaminated food products or direct contact with contaminated environmental factors, such as cat litterboxes [2]. Toxovax is currently the only market-approved toxoplasmosis vaccine available for use, but mutation risks and the short half-life of this vaccine have strictly prohibited its application in humans [3]. While a clinical toxoplasmosis vaccine is strongly desirable, its development is impeded by the challenges posed by the complex parasitic life cycle, host immune evasion and T. gondii strain diversity [4].

Various T. gondii antigens present in the secretory organelles have been tested as vaccine candidates, including the rhoptry proteins (ROP) which are of significant interest for their role in virulence and host cell invasion [5]. To date, many of these ROP antigens were incorporated as experimental DNA vaccines and their efficacies were evaluated in mice [6]. Among the ROP members, ROP18 was reported as one of the antigens that elicited the highest protection in mice [7]. However, it is worthwhile mentioning that the protection elicited by these DNA vaccines is suboptimal and lingering safety issues hinder their development [8]. Several different strategies could be utilized to overcome these challenges, with one of them being the application of heterologous immunization strategy which has been proven to be better inducer of protective immunity against toxoplasmosis than its homologous counterpart for several vaccine platforms [9,10]. Alternatively, a different vaccine platform that induces stronger immune responses than DNA vaccines could be used such as the virus-like particles (VLPs) expressing the ROP18 antigen that conferred partial protection against the highly virulent type I T. gondii lineage [11]. However, apart from our earlier ROP18 vaccine study [12], studies incorporating both heterologous immunization and VLPs are severely lacking and further assessing this immunization regimen could be beneficial for toxoplasmosis vaccine development.

Recently, we assessed the protection elicited by VLPs expressing the T. gondii CST1 antigen which was demonstrated to be highly efficacious [13]. Although tissue cyst formation in the brains of immunized mice were suppressed to remarkably low levels, the vaccine failed to offer complete protection as indicated by trace amounts of tissue cyst persistence. Given that recombinant baculovirus (rBV) and VLPs expressing the ROP18 antigen successfully induced protection against T. gondii ME49 in mice [12], we hypothesized that replacing the ROP18 antigen with the CST1 would likely induce equal, if not better protection. To test this hypothesis and also improve the overall efficacy of the CST1 VLPs, heterologous immunization strategy involving CST1-expressing rBV and VLP vaccine platforms were assessed. We also compared the overall efficacy to a well-characterized vaccine candidate antigen ROP18 using identical immunization approach to confirm the suitability of the CST1 for further vaccine development.

2. Materials & methods

2.1. Mice & ethics statement

Six-weeks-old female BALB/c mice were acquired from NARA Biotech (Seoul, South Korea). All procedures involving these animals were approved by and conducted in accordance with the ethical standards of the Kyung Hee University’s Institutional Animal Care and Use Committee (permit ID: KHSASP-21-250) and all efforts were made to minimize animal suffering. To prevent animals from exceeding the humane intervention end point, appropriate measures were implemented and mice reaching this point were humanely euthanized using a CO₂ chamber.

2.2. Generation of recombinant baculovirus (rBV) & VLPs

Genes for T. gondii ROP18 and CST1, and influenza M1 were PCR amplified and cloned in pFastBac vector. The recombinant plasmids were confirmed by DNA sequencing. Recombinant baculoviruses expressing the CST1 (CST1-rBV), ROP18 (ROP18-rBV) and M1 (M1-rBV) antigens were produced following the methods described in our earlier works [11,13]. M1-rBV, combined with either CST1-rBV or RO18-rBV, were co-transfected into Sf9 cells for CST1 and ROP18 VLP assembly, respectively. To characterize the self-assembled CST1 and ROP18 VLPs, western blotting was performed. Briefly, VLPs of varying concentrations were resolved using SDS-PAGE and subsequently transferred onto nitrocellulose membranes. These membranes were blocked with 5% skim milk prepared in Tris-buffered saline with 0.1% Tween-20 (TBST), followed by overnight incubation with T. gondii polyclonal antibodies (1: 500 dilution in TBST) or monoclonal M1 antibodies (1: 3000 dilution in TBST) at 4°C. Subsequent steps included washing, incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies, and visualization of protein bands using the ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA) with enhanced chemiluminescence. Both VLPs and rBVs were also directly visualized using transmission electron microscopy (TEM) by staining the samples placed on carbon-coated girds with uranyl acetate, and observing under the Bio-High voltage EM system.

2.3. Immunization & challenge infection

A total of 28 mice were randomly selected and subdivided into four different groups. Eight mice were immunized with CST1, and another eight mice were vaccinated with ROP18 antigens. Of the remaining 12 mice, eight were selected and grouped as the Naive + Challenge (NC) infection control and the remaining four mice were grouped as Naive (N) control. Mice belonging to the CST1, ROP18 and NC groups were intranasally primed with 4 × 10⁴ plaque forming units of CST1-rBV, ROP18-rBV and PBS, each respectively. Primed mice received two additional boost immunizations at 4-week intervals with either 50 μg of each VLPs or PBS via the intranasal (IN) route. Four weeks after the final vaccination, with the exception of the naive group, all of the mice were orally challenged with 450 T. gondii (ME49) cysts.

2.4. T. gondii-specific IgG & IgA response in sera

Enzyme-linked immunosorbent assays (ELISA) were performed to evaluate parasite-specific antibody responses. Briefly, 96-well plates were coated with 100 μl of carbonate coating buffer solution containing 5 μg/ml of T. gondii soluble total antigens overnight at 4°C as indicated [14]. Wells were rinsed with phosphate-buffered saline containing 0.05% Tween-20 (PBST) and incubated with 0.2% gelatin at 37°C, 2 h. Diluted mouse sera (1:50 dilution in PBST) were then added to the wells and incubated at 37°C for 1 h. After repeated washing, goat antimouse HRP-conjugated IgG or IgA secondary antibodies (Southern Biotech, Birmingham, AL, USA) were added to each well (1:2000 dilution in PBST) and plates were incubated for another 1 h, 37°C. Colorimetric assay was performed by dispensing 100 μl of citrate buffer containing o-phenylenediamine substrate and H₂O₂ into each well. Reactions were stopped by adding 2N H₂SO₄ and absorbance readings at 492 nm were measured using a microplate reader (Enzo Life Sciences, Farmingdale, NY, USA).

2.5. Antibody-secreting cell (ASC) responses

Spleens of sacrificed mice were harvested and homogenized to prepare single cell suspensions of splenocytes. ASC responses were determined following the previously described method [12,15]. In brief, a total of 1 × 10⁶ splenocytes immersed in complete RPMI-1640 media (Welgene, Gyeong-san, South Korea) with 10% fetal bovine serum and 1% penicillin/streptomycin were seeded into each well, and plates were incubated for 5 days at 37°C, 5% CO₂. After 5 days of incubation, supernatants were discarded and HRP-conjugated antimouse IgG or IgA antibodies were added to respective wells. Once o-phenylenediamine substrate dissolved in citrate buffer with H₂O₂ were added to the plates, reactions were stopped with diluted sulfuric acid and OD_492 nm readings were measured using a microplate reader.

2.6. B cells responses in spleen by flow cytometry

Splenocytes were processed for flow cytometric analysis as previously described [16]. Briefly, single cell suspensions of splenocytes were stimulated with T. gondii total soluble antigen (5 μg/ml) for 4 h at 37°C with 5% CO₂. After antigen stimulation, cells were stained with fluorophore conjugated GL7-PE (Cat. #561530) and B220-FITC (Cat. #553087) antibodies at 1:100 dilutions (BD Biosciences, Franklin Lakes, NJ, USA). Flow cytometry procedures were performed following the manufacturer's protocol. Stained samples were analyzed using the Accuri C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

2.7. Proinflammatory cytokine assays

Brain tissues were harvested from each mouse and homogenized in PBS. After centrifuging the tissue homogenates, supernatants were carefully collected and stored at -80°C until use. To evaluate the production of proinflammatory cytokines IFN-γ and IL-6, ELISA kits from BD Biosciences were used (BD Biosciences, Franklin Lakes, NJ, USA). Experiments were performed following the manufacturer’s instructions and standard curves were drawn to calculate the cytokine concentration.

2.8. Parasite burden, bodyweight & survival measurement

Brain tissue homogenates were used to determine the parasite burden. Homogenates were centrifuged and the pelleted cells were resuspended in 200 μl of PBS. Tissue cysts were manually counted under the microscope by placing 10 μl of the cyst suspension on a clean microscope slide glass. A cover slip was gently placed on top and slides were observed under the microscope. The total number of cysts counted from the 10 μl portion was multiplied by 20 for normalization and data were presented as cyst counts per mouse brain tissue. Homogenates of each mouse were counted three-times. Mice were monitored daily to record any changes in body weight and survival. At 35 days post infection (dpi), which was identified as the humane intervention point due to significant body weight loss exceeding 20% of the initial value in the infection control group, all of the mice were humanely euthanized.

2.9. Statistical analysis

All samples were processed on an individual basis and experiments were performed in triplicates. Statistical analyses were performed using the Prism 8 software (GraphPad Software, San Diego, CA, USA). One-way analysis of variance was performed with Tukey’s post hoc test. Survival data were analyzed using the Kaplan–Meier analysis with log-rank test. Data are presented as mean ± standard deviation and *p < 0.05 was considered statistically significant, which were indicated using asterisks.

3. Results

3.1. Production of rBVs for VLP assembly in Sf9 cells

Western blotting was performed to confirm successful assembly of the CST1 and ROP18 VLPs. CST1 and ROP18 antigens were detected using the polyclonal T. gondii antibody acquired from infection control mice, while the influenza M1 antigen was detected using the monoclonal anti-M1 antibody. CST1 with a size of 43 kDa and ROP18 at 61 kDa were detected from the respective VLPs (Figure 1A & Supplementary Figure S1A). M1 antigen expressions were also confirmed at 28 kDa from both CST1 and ROP18 VLPs (Supplementary Figure S1B), indicating successful assembly of the two VLP vaccines. Band densities of the three antigens were proportional to the protein loading concentration, with lower band density being observed at 10 μg than at 25 μg. Both VLPs and the rBVs used for VLP assembly were visualized under TEM (Figure 1B). Spherically shaped VLPs, with the dark protrusions indicative of the target antigens were observed. Recombinant baculoviruses, characterized by their distinct rod-shaped morphology, were also confirmed. Based on these observations, we ascertained that both rBVs and VLPs were properly assembled and suitable for subsequent immunization studies.

Figure 1.

Characterization of the virus-like particles and recombinant baculovirus. The antigen components of VLPs were analyzed using western blot (A). The membranes were probed with polyclonal mouse anti-Toxoplasma gondii sera or anti-M1 monoclonal antibody to confirm antigen expression within the VLPs. The morphologies of CST1-VLPs, ROP18-VLP, CST1-rBV and ROP18-rBV were examined using transmission electron microscopy (B).

VLP: Virus-like particles.

3.2. Heterologous immunization involving rBVs & VLPs elicited T. gondii-specific serum IgG & IgA antibody responses

All animal experiments were performed following the immunization and experimental schedule as illustrated (Figure 2A). Briefly, mice were primed with either CST1-rBV or ROP18-rBV through the IN routes. VLPs were administered through identical routes at 4-week intervals and sera were collected at regular intervals after each immunization. Four weeks after the final VLP immunization, mice were infected with T. gondii cysts (ME49) and protective efficacy was assessed. Serum IgG responses against the T. gondii antigens were also evaluated using the sera acquired after each immunization (Figure 2B). At prime, neither CST1 nor ROP18 rBVs elicited significant induction in parasite-specific antibody responses. However, following VLP boost immunization, significant increases were observed and this trend also continued after the second boost immunization. Identical trend was also observed for serum IgA (Figure 2C). Although near-basal levels of T. gondii-specific antibody responses occurred, significantly enhanced IgA levels were detected, thus signifying that this vaccination approach is capable of eliciting parasite-specific immune response.

Figure 2.

Experimental schedule and antibody responses in sera. The schedule of immunization, infection, immunological analysis and body weight and survival monitoring was scrupulously followed as described above (A). Images were created with BioRender.com. Sera (n = 4 for naive, n = 8 for experimental group) were collected from mice one weeks after each immunization to measure the antibody responses against T. gondii. Parasite specific IgG (B) and IgA (C) levels were assessed using ELISA. Data are presented as mean ± SD (**p < 0.01; ***p < 0.001).

SD: Standard deviation.

3.3. Heterologous immunization induced splenic ASC response

Spleens of mice were collected after T. gondii challenge infection and ASC responses were evaluated. Both CST1 and ROP18 VLPs elicited IgG ASC responses that were significantly greater than those of NC control group (Figure 3A). Of the two VLPs, CST1 VLPs induced noticeably greater levels of IgG ASC than ROP18 VLP. This phenomenon was also observed for splenic IgA ASC as well (Figure 3B). Both CST1 and ROP18 VLPs evoked splenic IgA responses that were substantially greater than the NC group, with the former of the two being induced to a significantly greater extent than the latter. The ASC response elicited by NC was marginally increased, but the changes were negligible when compared with the N control group.

Figure 3.

Antibody secreting cell response. Splenocytes from mice (n = 4 for naive, n = 8 for experimental groups) were collected at 35 dpi and cultured in 96-well plates coated with Toxoplasma gondii ME49 antigen. Plates were incubated for 5 days to evaluate T. gondii-specific IgG (A) and IgA (B) ASC responses. Absorbance readings at 492 nm were measured using a microplate reader. Data are presented as mean ± SD (**p < 0.01; ***p < 0.001).

SD: Standard deviation.

3.4. Heterologous immunization of the T. gondii vaccines enhanced GC B cell frequencies in the spleens of mice

Flow cytometry was performed using the single cell suspensions of splenocytes harvested from mice. Marginal increase in B220 ⁺ GL-7⁺ GC B cell frequency was observed for NC, though negligible when compared with the N control (Figure 4A). In contrast to this, markedly enhanced GC B cell frequencies were detected from the splenocytes of vaccinated mice. CST1 vaccines ensured significantly greater GC B cell frequencies than the NC, whereas the differences between ROP18 and NC were negligible. A representative scatter plot depicting GC B cell frequencies along with the cell events corresponding to GC B cell (B220⁺/GL7⁺) was provided (Figure 4B). Compared with the N control, ROP18 and CST1 vaccination nearly doubled the cell counts of GC B cells, whereas NC had negligible effect.

Figure 4.

Germinal center B cell response. Splenocyte samples (n = 4 for naive, n = 8 for experimental groups) were processed on an individual basis and stained with appropriate surface antibodies for flow cytometry assessment. Cells were acquired and gated to quantify frequencies of GC B cell populations (A). Representative scatter plots for each group, depicting gated GC B cells (B220⁺/GL-7⁺) were also provided (B). Data are presented as mean ± SD (**p < 0.01).

SD: Standard deviation.

3.5. Heterologous immunization reduced inflammatory cytokine responses in the brain

To confirm that vaccines adequately suppressed the production of the inflammatory cytokines in the brains, cytokine ELISA was performed. IFN-γ production was detected in all of the challenge-infected mice, irrespective of the immunization status (Figure 5A). While cytokine production remained at near-basal levels in the naive control group, a drastic increase in IFN-γ production was detected in the NC group. Immunizing mice with either CST1 or ROP18-based vaccines significantly reduced the IFN-γ concentrations in the brain homogenates of mice. Consistent with this finding, similar trend was observed for IL-6 (Figure 5B). Compared with the NC control, significant reductions in IL-6 was observed in immunized mice.

Figure 5.

Proinflammatory cytokine response. Brain tissues of mice (n = 4 for naive, n = 8 for experimental groups) infected with Toxoplasma gondii ME49 were harvested at 35 dpi to measure proinflammatory cytokine production. Supernatants from brain homogenates were analyzed to measure the levels of IFN-γ (A) and IL-6 (B). Tissue samples were collected individually, each performed in triplicates. Data are presented as mean ± SD (*p < 0.05; **p < 0.01).

SD: Standard deviation.

3.6. CST1 & ROP18 vaccines protected mice from succumbing to death

T. gondii cysts were counted from the brain tissues of mice to assess vaccine-mediated protection. Drastic increase in cyst burden was observed in the brains of NC mice. However, immunizing mice with either CST1 or ROP18 vaccines suppressed the formation of brain tissue cysts (Figure 6A). Of the two vaccines, heterologous CST1 vaccine immunization was more effective as indicated by the significantly lower cyst burden than ROP18 vaccine group. Changes in body weight also revealed that CST1 vaccines conferred better protection than ROP18 vaccines (Figure 6B). CST1 immunization resulted in minimal body weight fluctuations over the 40 days of infection period, whereas roughly 10% of body weight loss was observed in ROP18-immunized mice. Gradual and continuous weight loss was only detected from the NC control group. Significant differences in body weight was observed at 30 and 35 dpi between CST1 and NC. On the contrary, weight differences between ROP18 and NC were negligible at all time points. Vaccination ensured that all of the mice survived, confirming that their inoculation induced protection (Figure 6C). We found that vaccination with either ROP18 or CST1 was positively correlated with the overall survival of mice. While 100% survival was observed for both CST1 and ROP18, survival in the NC group steadily declined and body weights of three mice eventually reached the humane intervention point.

Figure 6.

Protection upon challenge infection with Toxoplasma gondii ME49. BALB/c mice (n = 4 for naive, n = 8 for experimental groups) were orally infected with a lethal dose of T. gondii four weeks after last immunization. At 35 dpi, brain tissues (n = 4) were harvested to count cysts isolated from the homogenates. Cysts were counted under the microscope from multiple fields of view per sample (A). Mice were monitored daily to assess changes in body weight (B) and survival (C) after T. gondii ME49 infection. Data are presented as mean ± SD (*p < 0.05; ***p < 0.001).

SD: Standard deviation.

4. Discussion

Here, we evaluated the efficacy of the heterologous immunization strategy involving rBV and VLP vaccines against toxoplasmosis. Our findings showed that heterologous immunization with vaccines expressing the CST1 antigen was more efficacious than those expressing the ROP18 antigen. Previously, several research groups have reported that baculovirus-derived vaccines can be used to confer protection against various parasitic infections, including Trypanosoma cruzi, Schistosoma mansoni, Neospora caninum and Plasmodium vivax [17–20]. Similarly, mice immunized with VLPs displaying a wide array of T. gondii antigens were well-protected and none of the vaccinated mice perished upon T. gondii infection [13,21]. In line with these previous reports, we have previously demonstrated that heterologous immunization with ROP18-rBV and ROP18-VLP vaccines induce protection against T. gondii ME49 challenge infection in mice [12]. Based on these findings which clearly illustrated the protective efficacy of both rBV and VLP-based vaccines, we anticipated that the heterologous immunization strategy employed here would confer adequate protection in mice. As expected, the heterologous immunization strategy involving rBV and VLP vaccine platforms ensured 100% survival of immunized mice and significantly reduced their parasite burden.

To our surprise, CST1-based vaccines were more efficacious than the ROP18-expressing vaccines, especially when considering the fact that both CST1 and ROP18 antigens are associated with the bradyzoite stage of the parasite's life cycle [22,23]. Even the differences in inflammatory cytokines induced upon challenge infection were negligible for the two vaccination groups. Specifically, cytokines such as IFN-γ and IL-6 are associated with bradyzoite differentiation that leads to the formation of cysts. Earlier studies delineated that increasing IL-6 levels enhances cyst formation during the acute stage of T. gondii infection and numerous accounts of how IFN-γ regulates bradyzoite differentiation have been documented [24–26]. The exact causation leading to these discrepant findings remain unknown and would be worthwhile investigating. Though speculative, one possible explanation could be the antigen polymorphism in ROP18 [27]. Such genetic heterogeneity observed across different T. gondii genotypes for the ROP18 antigen indicates that a highly specific and purified vaccine component may fail to elicit less than optimal protection [28,29]. CST1, unlike the ROP18 antigen, could be highly conserved across the T. gondii lineages and this may have led to slightly better vaccine efficacy detected in the present study. In support of this notion, the large mucin domain of the T. gondii CST1 was reported to resembled those observed from a phylogenetically distant Apicomplexan parasite such as Cryptosporidium parvum [22,30,31].

Despite the results obtained for the CST1-expressing vaccines, several inherent limitations of this study must be addressed. It is widely accepted that healthy individuals are often asymptomatic when infected with T. gondii whereas symptoms can be severe for immunocompromised individuals [32]. BALB/c mice are more resilient to T. gondii infection than C57BL/6 mice and as such, vaccine studies should be conducted in a more susceptible animal model to test the true protective efficacy of the vaccines and the immunization strategy demonstrated here [33,34]. Furthermore, the longevity of the immune response elicited by these vaccines also remains an area of uncertainty. Understanding the duration and memory capacity of the immune response elicited by this heterologous immunization strategy is crucial, particularly in the context of developing a vaccine that offers long-term protection.

5. Conclusion

In summary, we unveiled the potential of a heterologous immunization strategy which incorporated recombinant baculoviruses and VLPs. The protective efficacy of CST1 or ROP18-expressing vaccines were compared and while both elicited immune responses, mice were better protected against T. gondii ME49 challenge infection by CST1-based vaccines. Our findings suggest that the CST1 antigen could play a pivotal role in developing an effective vaccine against toxoplasmosis, offering a promising pathway toward mitigating this global health challenge.

Funding Statement

This study was supported by the Core Research Institute (CRI) Program, the Basic Science Research Program through the National Research Foundation of Korea (NRF), Ministry of Education (NRF2018R1A6A1A03025124).

Supplemental material

Supplemental data for this article can be accessed at https://doi.org/10.1080/17435889.2024.2403333

Author contributions

Conceptualization: F-S Quan; Formal analysis: G-D Eom; Funding acquisition: SS Kim; Investigation: G-D Eom, KB Chu, J Mao, K-W Yoon, H-J Kang; Methodlogy: F-S Quan; Project Administration: SS Kim, F-S Quan; Resources: E-K Moon; Supervision: F-S Quan; Validation: G-D Eom, KB Chu, K-W Yoon; Visualization: G-D Eom; Writing – original draft: G-D Eom, KB Chu; Writing – review & editing: KB Chu, F-S Quan.

Financial disclosure

This study was supported by the Core Research Institute (CRI) Program, the Basic Science Research Program through the National Research Foundation of Korea (NRF), Ministry of Education (NRF2018R1A6A1A03025124). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

All procedures involving these animals were approved by and conducted in accordance with the ethical standards of the Kyung Hee University's Institutional Animal Care and Use Committee (permit ID: KHSASP-21-250) and all efforts were made to minimize animal suffering.