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Microbes & Immunity Carotene and immunity to COVID-19 vaccine

The gastrointestinal tract (GIT) is the home to the gut- 2. Materials and methods
associated lymphoid tissue (GALT), the largest immune
organ in the human body. GALT forms part of the mucosa- 2.1. Materials
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associated lymphoid tissue and comprises many immune CaroGaia (30% oil suspension) used for dietary
cells. Hence, GIT is a primary site for immune activities. At intervention was a gift from PhytoGaia Sdn Bhd, Malaysia.
the same time, the GIT, particularly the colon, serves as an The vehicle used was palm oil (Buruh Cooking Oil, Lam
invaluable repository for gut microbiota, such as bacteria, Soon, Malaysia). The inactivated SARS-CoV-2 vaccine
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viruses, and fungi. The comprehensive collection of gut (CoronaVac, Sinovac Biotech Ltd, China) was a kind gift
microbiomes can contribute to a vast palette of metabolic from Pharmaniaga Lifescience Sdn Bhd, Malaysia.
enzymes and pose a greater metabolic potential than
their host. Due to the proximity between gut microbiota 2.2. Ethical approval
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and GALT, they inevitably affect one another as these The ethical approval for this study was obtained from
gastrointestinal inhabitants can interact with nutrition and the Monash University Animal Ethics Committee (AEC
selectively promote or inhibit specific bacteria, thereby Project ID: 29899).
modulating gut microbiota populations. In addition, the
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gut microbiome plays crucial roles in nutrition metabolism, 2.3. Animal study design
energy metabolism, and immunomodulation. Studies Ten-week-old male BALB/c mice were obtained and
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reported that short-chain fatty acids (SCFAs), metabolites housed individually at the animal holding facility of
produced by the gut microbiota, can enhance B-cell Monash University Malaysia. A total of 32 mice were used
metabolism to support the production of the energy in this study. The mice were acclimated to the housing
required to generate antibodies and cytokines. These conditions for 1 week before the study began. During the
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metabolites also upregulate genes responsible for plasma study, the mice were subjected to a 12-h light/12-h dark
cell differentiation and class switching, vital for immune cycle with ad libitum access to food and water. A standard
responses to pathogens or vaccines. The present literature mouse pellet chow was provided throughout the study.
suggests that carotene supplementation can modulate host The experimental design is illustrated in Figure 1. Briefly,
gut microbiota and SCFA production. 32 male BALB/c mice were evenly divided into two dietary
The SCFAs are key metabolites produced by gut intervention groups, that is, (i) control (fed with the vehicle
microbiota, which can modulate immune response and – palm oil) and (ii) carotene-fed group. The mice were fed
regulate the activity of innate cells, such as macrophages, daily with 100 µL of carotene (50 mg/kg body weight) or
neutrophils, and dendritic cells. For example, SCFAs were palm oil (vehicle) through gavage. On day 14, the mice in
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reported to limit SARS-CoV-2 infection by downregulating each dietary group were split into two sub-groups, that
the level of angiotensin-converting enzyme 2, the main is, (i) non-vaccinated (phosphate-buffered saline [PBS])
receptor responsible for viral infections, while improving and (ii) vaccinated (CoronaVac) (Table 1). Vaccination
antiviral immunity. In addition, SCFAs can also control was administered by an intramuscular injection into the
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SARS-CoV-2 infection by downregulating transcription hind leg of the mice using a 30G needle, with 0.1 mL
of genes responsible for modulation of viral entry and of CoronaVac (0.6 µg) as vaccine intervention for the
replication, such as IFN, transmembrane protease vaccinated subgroup and 0.1 mL of PBS as control for the
serine 2, and retinoic acid-inducible gene 1 receptors, non-vaccinated subgroup. On day 42, plasma samples were
while maintaining the permeability of intestinal cells. collected from the mice and were given a second dose of
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Moreover, SCFAs can modulate the differentiation of T CoronaVac or PBS. At the study’s endpoint (day 70), the
lymphocytes and B cells, which are responsible for the mice were humanely euthanized, and samples of plasma,
adaptive immune response. 22 feces, and spleens were collected for analysis.
This study used a mouse model to investigate how daily
carotene supplementation modulates the immune response 2.4. Mice euthanasia
to COVID-19 vaccination. This research aimed to unveil On day 70, the mice were weighed to calculate and
the mechanism of action of carotene supplementation in prepare the dosage for a ketamine and xylazine cocktail,
immune response post-vaccination and provide evidence at concentrations of 100 mg/kg and 10 mg/kg, respectively.
of its health benefits. In addition, this study also explored The concoction was administered through intraperitoneal
how carotene influences gut microbiota and SCFA injection. Once the mice were determined to be fully
production, offering insights into the overall action of anesthetized, euthanasia was performed by cardiac
carotene supplementation. puncture.

Volume 2 Issue 3 (2025) 74 doi: 10.36922/MI025110021