Medical application of reuterin in preparation of vaccine adjuvant

Abstract

The invention provides an application of reuterin in preparation of a vaccine adjuvant, and provides a novel vaccine adjuvant which is reuterin which is a substance with an immunocompetence inducing and domesticating and is generated by lactobacillus reuteri. Compared with a classical aluminum adjuvant, the reuterin can induce a remarkable cellular immune response and the generation of a high-affinity antigen-specific antibody, and when the reuterin is combined with the aluminum adjuvant for use, the acute inflammatory response caused by the aluminum adjuvant is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccine preparation, and in particular provides the use of reuterin in the preparation of vaccine adjuvants, and discloses a new medical use of reuterin. Background Art

[0002] Vaccines are effective means for preventing and controlling infectious diseases. A vaccine adjuvant refers to a substance that enhances the immunogenicity of an antigen through non-specific mechanisms, and it can promote, prolong or enhance the specific immune response to the vaccine antigen. As an important component of vaccines, adjuvants significantly improve the immune efficacy of vaccines by enhancing the immune response, and have become a key link in vaccine research and development. In recent years, significant progress has been made in the research of vaccine adjuvants, especially in enhancing immune responses, prolonging immune memory and improving vaccine effects. However, existing vaccine adjuvants still have some limitations, such as the potential to cause side effects, excessive immune responses or imbalanced immune responses, which limit their widespread application. With the rapid development of DNA recombinant technology, the research and development of new vaccines (such as recombinant subunit vaccines, synthetic peptide vaccines and nucleic acid vaccines) have also made significant progress. However, due to the small antigen molecular weight and weak immunogenicity of these vaccines, adjuvants are usually required to enhance the immune effect. Microorganisms and their metabolites have become important vaccine adjuvants because of their easy availability, low cost and ability to effectively stimulate immune responses.

[0003] Reuterin is a water-soluble small molecule antibacterial compound produced by the probiotic Lactobacillus reuteri ( Limosilactobacillus reuteri ), and has attracted much attention in recent years. As a small molecule with broad-spectrum antibacterial activity, reuterin can not only inhibit a variety of pathogenic microorganisms, but also play an important role in maintaining the balance of the intestinal microbiota, regulating immune responses and promoting intestinal health. In addition, as a natural antibacterial substance, reuterin has broad application prospects in the fields of food, health products and clinical medicine. Summary of the Invention

[0004] The present invention discloses the use of reuterin in the preparation of vaccine adjuvants, provides a new use of reuterin, and can reduce the acute inflammatory response caused by aluminum adjuvants, and has good effects.

[0005] Through the analysis of mouse in vitro peritoneal macrophage models and bone marrow-derived macrophage models, the present invention verified the immune response induced by reuterin. The in vivo adjuvant effect analysis of mice showed that reuterin can be used as a vaccine adjuvant to enhance the immune response of the model antigen OVA and has the ability to induce cellular immune responses.

[0006] The positive effects of the present invention are as follows: A new vaccine adjuvant is provided, which is reuterin produced by Lactobacillus reuteri and has the activity of inducing trained immunity; compared with the classical aluminum adjuvant, reuterin can induce significant cellular immune responses and the production of high-affinity antigen-specific antibodies, and when used in combination with the aluminum adjuvant, it reduces the acute inflammatory response caused by the aluminum adjuvant. Description of the Drawings

[0007] Figure 1 For reuterin to induce the trained immune response of macrophages in vitro; Figure 2 For reuterin to induce the enhancement of macrophage functional activity by trained immunity; Figure 3 For reuterin to induce the trained immune response of mouse bone marrow macrophages; Figure 4 For reuterin to induce the trained immune response in mice; Figure 5 For reuterin to enhance the immune response to antigen OVA; Figure 6 For reuterin to enhance the analysis of OVA antibody subclasses; Figure 7 For the effect of reuterin immunization on spleen lymphocyte subsets; Figure 8 For the safety evaluation after reuterin immunization; Figure 9 For reuterin to inhibit the inflammatory response induced by LPS. Detailed Embodiments

[0008] The present invention will be further illustrated by some specific embodiments below. It should be clear that the embodiments described below are part of the embodiments of the present invention, rather than all of them, but the present invention is not limited to the scope of the described embodiments for this reason. The experimental methods without specific conditions in the following embodiments are carried out according to conventional methods or according to the product specifications.

[0009] Example 1. Verification of the trained immune response induced by reuterin using an in vitro peritoneal macrophage model

[0010] The trained induction activity of reuterin was analyzed using an in vitro trained immune model of mouse peritoneal macrophages. The specific method was as follows: As shown in Figure 1 A, first, mouse peritoneal macrophages were isolated, and heat-killed Candida albicans (HK. C. aAs a positive control, after stimulating macrophages for 24 h, the cells were washed twice with PBS to remove the stimulant, and then the cells were allowed to rest for 5 d. Finally, the cells were restimulated with 100 ng / mL of LPS or 1 μg / mL of Pam3CSK4. After 24 h, the levels of nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in the cell culture supernatant were detected. As Figure 1 shown in B-D, reuterin-conditioned macrophages significantly enhanced the immune response after restimulation, as evidenced by a significant increase in NO production and TNF-α levels, even exceeding that of HK. C. a In addition, after restimulation with Pam3CSK4 (TLR1 / 2 ligand), reuterin-conditioned macrophages significantly induced the production of TNF-α. The increased secretion level of IL-6 further confirmed this phenomenon. The present invention further explored whether glycolytic reprogramming was involved in the reuterin-induced trained immunity response. The results were as Figure 1 shown in E-F. The addition of the glycolysis inhibitor 2-deoxy-D-glucose (2-DG) before reuterin pre-stimulation did not significantly affect the secretion of TNF-α by macrophages, indicating that glycolysis was not crucial for this process. In contrast, the methyltransferase inhibitor 5′-deoxy-5′-(methylthio)adenosine (MTA) significantly inhibited the reuterin-induced trained immunity response. These results suggest that reuterin mainly induces trained immunity through epigenetic mechanisms.

[0011] To further explore the effect of reuterin on macrophage function, the present invention evaluated the phagocytosis and killing ability of macrophages. As Figure 2 shown in A-B, compared with unconditioned macrophages, the ability of reuterin-conditioned macrophages to phagocytose Staphylococcus aureus was significantly enhanced. In addition, the bacterial killing experiment showed that the killing ability of reuterin-conditioned macrophages against Staphylococcus aureus was also significantly improved. To evaluate the function of reuterin-conditioned macrophages in the tumor microenvironment, the present invention used the culture supernatant of B16-F10 melanoma cells to stimulate macrophages. The results were as Figure 2 shown in C-F. Conditioned macrophages secreted more NO, ROS, and TNF-α. In the experiment of co-culturing with tumor cells, conditioned macrophages exhibited enhanced tumor cell killing ability. Therefore, reuterin enhanced the phagocytosis and killing activities of macrophages, thereby improving their functions in anti-infection and anti-tumor immunity.

[0012] Example 2. Analysis of the trained induction activity of reuterin in mouse bone marrow-derived macrophages

[0013] To verify the universality of reuterin-induced trained immunity in macrophages, the present invention further conducted experiments using bone marrow-derived macrophages. The results were as Figure 3As shown in A-B, compared with the macrophages of the non-primed group, the macrophages of mice treated with reuterin showed a significant increase in NO release and TNF-α secretion after LPS restimulation. Therefore, reuterin can induce trained immunity in macrophages from different sources.

[0014] Example 3: Trained immune response induced by reuterin in mice

[0015] To further verify whether reuterin can induce trained immunity in vivo, the present invention conducted in vivo experiments. As Figure 4 shown in A, after mice were pre-exposed to reuterin, the immune response of the isolated macrophages to LPS restimulation was significantly enhanced. The results are as Figure 4 shown in B-D, specifically manifested as an increase in the levels of NO and TNF-α. In addition, the mRNA expression of the inflammatory cytokines IL-6 and IL-1β was also significantly upregulated, further supporting the biological activity of reuterin in inducing trained immunity in vivo.

[0016] Example 4: Analysis of the adjuvant effect of reuterin

[0017] To evaluate the adjuvant activity of reuterin, as Figure 5 shown in A, the present invention subcutaneously immunized with ovalbumin (OVA) as a model antigen and evaluated the level of OVA-specific antibodies after immunization. The results are as Figure 5 shown in B-C, the OVA-specific IgG titer of the mice immunized with OVA + reuterin (100 μg) was significantly higher than that of the group immunized with OVA alone, although it was lower than that of the traditional aluminum adjuvant group. However, when reuterin was used in combination with aluminum adjuvant, the highest IgG response was shown. The results of antibody affinity evaluation indicated that reuterin was superior to aluminum adjuvant in improving the quality of antibodies. In addition, the present invention also evaluated the levels of IgG1 and IgG2c subclasses after immunization. The results are as Figure 6 shown in A-B, compared with the use of OVA or reuterin alone, aluminum adjuvant significantly induced a stronger IgG1 response, while the highest IgG1 response was shown when reuterin was used in combination with aluminum adjuvant. In contrast, the OVA + reuterin group promoted a higher level of IgG2c production, indicating that the immune response induced by reuterin was biased towards Th1, while the Th2 response was insufficient. The OVA-specific memory lymphocyte response is as Figure 6 shown in C, the reuterin adjuvant group significantly promoted the proliferation of antigen-specific lymphocytes in the spleen, indicating that reuterin can effectively induce the formation of immune memory. The effect of reuterin on the cellular immune response was further evaluated, and the results are as Figure 7 shown in A-B, compared with the mice immunized with OVA alone, aluminum adjuvant significantly increased the proportion of CD4 + T cells, while the reuterin group showed a CD4+ A slight increase in the proportion of T cells. In contrast, aluminum adjuvant has less effect on the proportion of CD8 + T cells, while reuterin significantly increases the proportion of CD8 + T cells, suggesting that reuterin may promote cell-mediated immune responses by enhancing the Th1-biased response. Therefore, the combined use of the two can induce a more balanced immune response. The safety of reuterin vaccination was further evaluated, and the results are as follows Figure 8 Shown in A - B, compared with the use of aluminum adjuvant alone, the combined use of reuterin and aluminum adjuvant significantly reduced the TNF-α level induced by aluminum adjuvant. In addition, no significant weight change was observed between groups during the whole experiment, further supporting its good tolerance. The results of cell experiments are as follows Figure 9 Shown in A - B, it was found in the present invention that LPS treatment of macrophages significantly increased the concentrations of NO and TNF-α. However, when reuterin was combined with LPS treatment, the concentrations of NO and TNF-α decreased significantly. These results indicate that reuterin has potential anti-inflammatory effects.

[0018] As can be seen from the above embodiments, the present invention provides a natural immune memory-inducing active substance - reuterin, and the natural immune memory-inducing activity, adjuvant activity and safety of reuterin were evaluated. However, it can also be seen that based on the present invention, some modifications and expansions can be made. Therefore, modifications or improvements made without departing from the spirit and principles of the present invention fall within the scope of protection required by the present invention.

Claims

1. Use of reuterin in the preparation of vaccine adjuvants.

2. The use according to claim 1, characterized in that, The adjuvant effect of reuterin is based on its biological activity that enhances the induced trained immune response.

3. The use according to claim 1, characterized in that, The application includes inducing the body to produce specific high-affinity antibodies and cellular immune responses.

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