Several clinical studies have demonstrated the efficacy of split-virus vaccines20,21, but the effect is not complete and further improvement is strongly demanded. Adequate innate immune signaling is essential to elicit acquired immune response22. However, there are not adequate PRR ligands in the split-virus and subunit vaccines. A recent study suggested that TLR5, a flagellin receptor, compensates for the innate immune stimuli through the microbiome3. Considering the importance of innate immune responses in the development of acquired immunity lacking in split-virus vaccines, TLR5 may be choke point in the split virus vaccine sensing. TLR5 knockout mice showed a drastically low sensitivity toward the split vaccine-elicited immune response but were responsive to live vaccines (that containing PAMPs)3. Consistent with these findings, TLR5 expression correlated with HAI titer in influenza split vaccine-treated humans4. However, no applicable approach has been established to upregulate TLR5 expression.
Here, we show for the first time that EGCG3”Me, the characteristic polyphenol in green tea cultivar Benifuuki, upregulated TLR5 expression on splenocytes, macrophages, intestines, and LPDCs, and enhanced the effect of the split vaccine in a mouse model. To the best of our knowledge, EGCG3”Me is the first orally delivered exogenous compound to upregulate TLR5 expression. EGCG3”Me enhanced the split-virus vaccine-induced HAI titer, a criterion used to evaluate protective effects of vaccines13.
In the prevention strategy, a risk/benefit balance is crucial. This tea cultivar is commercially available, widely consumed for a long time, economic, and safe, making it a potential supplement to boost split-virus vaccine efficacy. EGCG3”Me enhanced the split vaccine-elicited immune response accompanied with upregulating TLR5 expression. Moreover, green tea has no alcohol, sugar, or salt, and is consumed in many parts of the world. Considering these characteristics, Benifuuki could be a potent candidate to enhance the effect of split-virus vaccines.
The HED for EGCG3”Me (20 mg/kg body weight in mouse; split vaccine model) is 1.62 mg/kg body weight, equating to 97.2 mg/person (60 kg). This is amount of EGCG3”Me contained in 6 g green tea leaves7,8 and could be applicable in our daily life.
The microbiome is a hot topic in the healthcare industry. The microbiome has strong effects on obesity23, immunity24, vaccine efficacy25, and immune checkpoint inhibitors in cancer26. Many probiotics and prebiotics have been developed and are under development; however, the regulation of the sensing mechanisms is not well elucidated. Considering that TLR5 is a crucial PRR to “sense” the microbiome27,28,29, EGCG3”Me could serve as a potent enhancer for these probiotics and prebiotics because its long-term safety has been validated30. Our results provide a molecular basis for the development of novel probiotics and prebiotics.
We reported that EGCG3”Me exerts anti-inflammatory31, anti-obesity9, and anti-allergy effects32. The effect of EGCG3”Me on TLR5 seems paradoxical. O’Mahony D.S. et al. recently demonstrated that pro-inflammatory cytokines IFN-γ and granulocyte macrophage colony-stimulating factor (GM-CSF) upregulate TLR2 and TLR4 expression on monocytes but suppress TLR5 expression33. Considering the negative action of EGCG3”Me on TLR4-dependent signaling in macrophages31, these effects may be involved in the upregulation of TLR5.
A recent study indicated that protein kinase C (PKC) deltaser664 phosphorylation plays a crucial role in the expression of TLR534. PKC inhibition downregulated TLR5 expression in HT29 cells. EGCG3”Me exhibits strong 67LR agonist activity9,35, and 67LR agonist phosphorylated PKC deltaser664 not only in multiple myeloma cells and chronic lymphoid leukemia36,37, but also in chronic myeloid leukemia38 and acute myeloid leukemia cells39. Considering that EGCG3”Me has favorable pharmacokinetic properties (five times higher area under the curve than EGCG8), these mechanisms also may contribute to the upregulation of TLR5 expression and warrant further studies.
Our ex vivo studies showed that EGCG3”Me upregulated TLR5 and IL-6 expression without affecting TNF-α expression (data not shown). EGCG3”Me exerts anti-inflammatory effects, but how it elicits IL-6 expression without PAMPs remains unclear. Recently, TLR5 has been identified as a receptor specific for damage-associated molecular patterns (DAMPs)40. However, the exact molecule is yet unknown. EGCG3”Me could possibly elicit IL-6 expression through the upregulation of TLR5 and increase the sensitivity to DAMPs.
We have previously reported that methylated EGCG retains its 67LR agonist activity31 and shows a superior pharmacokinetic character8. High absorbance and retention of 67LR agonist properties may contribute to the effects of methylated EGCG.
Previous studies have shown that green tea-inactivated influenza virus induced neutralizing antibodies against the virus41. Considering in our daily lives, these events are also involved in the influenza-prevention effect of green tea. Those studies have shown the direct interaction of EGCG and viruses, suggesting the increased effect of EGCG-treated viruses or EGCG-treated vaccine41. In this study, our setting was based on the potentiation of conventional vaccines. EGCG3”Me was administrated orally though the diet, while the split vaccine was administrated though injection.
Since we have previously reported the enhanced effect of Benifuuki (methylated EGCG containing green tea extract) on the split vaccine-induced immune response5, we focused on the effect of methylated EGCG on split vaccine-treated mouse in the present study. The antibody titer upregulating effect in EGCG3”Me and split vaccine in combination is drastic. The drug interaction between the EGCG3”Me and split vaccine is unclear because, to reveal the synergism, an isobologram analysis or combination index calculation is necessary, requiring many mice; this could not be performed due to ethical reasons. The limitation of our present study is that whether methylated EGCG had an additive or synergistic effect has not been determined. However, because a split vaccine consists of a viral antigen, methylated EGCG may enhance the antigen-elicited immune response.
Recent research has shown that the microbiome can regulate host immunes system functions3. Considering the importance of immune regulation in the microbiome3, the effect of EGCG3”Me on the immune system could be mediated by its effects on the microbiome. Previous reports have also suggested that TLR5 expression can regulate the microbiome42. Considering that EGCG3”Me upregulates TLR5 expression in the intestine, these processes are complicated and possibly interconnected. EGCG3”Me induces TLR5 expression in vitro, and this direct action may contribute to its effect. In conclusion, EGCG3”Me, the characteristic polyphenol in Benifuuki, increased TLR5 expression on macrophages and LPDCs in split vaccine-administrated mice, and enhanced the vaccine-elicited immune response in vivo. Considering that EGCG3”Me can be consumed through green tea cultivar Benifuuki, EGCG3”Me may be a good candidate to improve performance of split vaccine. Because TLR5 acts as a sensing molecule in the microbiome, EGCG3”Me could be a potent approach to increase the sensitivity of prebiotics and probiotics, thus demanding further clinical research.
