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Microbes & Immunity Carotene and immunity to COVID-19 vaccine

Figure 1. The animal study design. In the supplementation period, half the mice were fed daily with 100 µL vehicle (palm oil), while the other half
were fed with 100 µL carotene (50 mg/kg body weight) by oral gavage for 70 days. For vaccination, half the mice in each supplementation arm received
intramuscular injections of 100 µL (0.6 µg) of the CoronaVac or 100 µL PBS (control) on days 14 and 42.

Table 1. Animal grouping 2.7. Immunophenotyping using flow cytometry
Dietary intervention Vaccine intervention Number of The peripheral blood lymphocytes collected in heparinized
mice tubes were analyzed using fluorochrome-labeled antibodies
+
+
+
Vehicle (palm oil) PBS 8 to CD3 T lymphocytes, CD4 Th cells, CD8 CTLs, and B
C19V 8 cells, and a flow cytometer (FACSVerse cell analyzer, BD,
Carotene (50 mg/kg body weight) PBS 8 USA). The immunostaining was carried out according to
the manufacturer’s protocol.
C19V 8
Total 32 2.8. IFN-γ production
Abbreviations: C19V: CoronaVac vaccine; PBS: Phosphate-buffered The amount of IFN-γ, a cytokine crucial for macrophage
saline.
activation and antigen presentation, in the supernatant of
splenocyte cultures, was quantified using a commercial
2.5. Isolation of splenocyte mouse IFN-γ enzyme-linked immunosorbent assay
During the autopsy, the spleen was aseptically collected and (ELISA) kit (#88-7314-88, Invitrogen, USA) using the
transferred to a petri dish containing 6 mL of RPMI 1640 manufacturer-recommended protocol.
culture medium (11875-093, Gibco, USA) supplemented
with 5% fetal bovine serum (FBS; A5256701, Gibco, USA), 2.9. Plasma SARS-CoV-2 recombinant receptor-
1% penicillin-streptomycin (15140122, Gibco, USA), and binding-domain antibody levels
1% glutamine. The splenic capsules were carefully snipped The plasma SARS-CoV-2 recombinant receptor-binding-
to release the splenocytes into the medium. The resulting domain (RBD) antibody level was quantified using the
splenocyte suspension was transferred into 15 mL Falcon SARS-CoV-2 RBD mouse immunoglobulin G (IgG)
tubes and kept on ice. Splenocytes were isolated through ELISA kit (CoV2-RBD mIgG, Novatein Biosciences, USA)
centrifugation (1,200 rpm for 10 min at 4°C). as per the manufacturer-recommended protocol. A mouse
SARS-CoV-2 spike-neutralizing antibody (#40592-MM57,
2.6. Splenocyte proliferation assay Sino Biological, China) was used as a positive control.
This assay was carried out to determine the proliferation The assay was carried out according to the recommended
of splenocytes in response to stimulation with CoronaVac. protocol.
The supernatant was discarded, and the splenocytes were 2.10. Characterization of gut microbiota
resuspended in 1 mL of complete culture medium (RPMI
1640 medium supplemented with 10% FBS and penicillin- 2.10.1. DNA purification and sequencing
streptomycin). The cell density was adjusted to 5 × 10⁴ To evaluate the impact of carotene supplementation on
splenocytes/mL in complete medium containing 10 µg/mL gut microbiota, 16S amplicon sequencing was performed
of COVID-19 vaccine. The cells were seeded into a 96-well on fecal samples collected at the endpoint, day 70. The
culture plate at 200 µL per well (5 × 10³ cells/well), and bacterial genomic DNA (gDNA) was extracted using
the plate was incubated for 72 h at 37°C in a humidified the QIAamp® PowerFecal® Pro kit (QIAGEN, Germany)
incubator with 5% CO₂. Splenocyte proliferation was according to the manufacturer’s instructions. The
assessed using the Cell Counting Kit-8 (CK04-01, Dojindo, quality and concentration of the gDNA were determined
Japan). The culture supernatants were harvested and stored using a nano-spectrophotometer. The V3–V4 region
at -80°C for subsequent cytokine analysis. was targeted for sequencing, using forward primer

Volume 2 Issue 3 (2025) 75 doi: 10.36922/MI025110021