Application of compound 101-mer in preparation of influenza vaccine adjuvant and influenza vaccine

Abstract

The invention relates to application of a compound 101-mer in preparation of an influenza vaccine adjuvant and an influenza vaccine, and belongs to the technical field of biology. The compound 101-mer is used as an influenza vaccine adjuvant for preparing an influenza vaccine, the immune protection effect of the influenza vaccine can be improved, the specific total IgG antibody titer of an influenza antigen and IgG1, IgG2a, IgG2b and IgG3 subtype antibody levels are improved, complete protection is provided after challenge, tissue damage is relieved, and the antiviral activity is remarkably superior to that of compounds 18-mer, 19-mer, 27-mer and LPS; as an adjuvant, the compound 101-mer does not have observable toxicity, can still be well tolerated under a high dosage, and has relatively high safety, and the safety of the compound 101-mer is remarkably superior to that of LPS (Lipopolysaccharide); the action mechanism is clear, and the compound can be used as a Dectin-2 agonist to activate a host immune system through a Syk / NF-kappa B signal channel.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to application of a compound 101-mer in preparation of an influenza vaccine adjuvant and an influenza vaccine. BACKGROUND

[0002] An adjuvant is a kind of non-specific immune enhancer capable of enhancing or regulating the immune response of the body to an antigen, and can significantly improve the immunogenicity of the antigen or change the type of immune response. There are many kinds of adjuvants, such as aluminum salts (such as aluminum hydroxide and alum), bacterial-derived components (such as lipopolysaccharide), and cytokines. Among them, natural glycolipid adjuvants (such as LPS) are one of the commonly used vaccine adjuvants, which can significantly activate the immune system, but their severe toxicity and structural complexity limit clinical application. However, the development of existing fully synthetic glycolipid adjuvants is difficult, and there are many problems, including low purity, structural heterogeneity, batch instability, and poor immune protection effect, and there is no report on the application of fully synthetic glycolipid adjuvants to influenza vaccine adjuvants, which provides an important innovation space for the research in this direction. SUMMARY

[0003] Therefore, the purpose of the present application is to provide application of a compound 101-mer in preparation of an influenza vaccine, which can significantly enhance the immune protection effect of the influenza vaccine and has good safety.

[0004] The present application provides application of a compound 101-mer in preparation of an influenza vaccine Preferably, the compound 101-mer is used as an adjuvant in the influenza vaccine.

[0005] Preferably, the compound 101-mer can enhance the immune protection effect of the influenza vaccine.

[0006] Preferably, the compound 101-mer can be used as a Dectin-2 agonist to activate the host immune system through the Syk / NF-κB signaling pathway.

[0007] The present application provides application of a compound 101-mer in preparation of an influenza vaccine adjuvant.

[0008] The present application also provides an influenza vaccine using the compound 101-mer as an adjuvant.

[0009] Preferably, the mass ratio of the antigen to the adjuvant in the influenza vaccine is (1:1) to (1:5).

[0010] Preferably, the antigen is an influenza virus hemagglutinin.

[0011] Preferably, the influenza is influenza A.

[0012] Preferably, the influenza is caused by the H1N1 influenza virus.

[0013] The beneficial effects of this invention are: This invention utilizes compound 101-mer as an adjuvant in influenza vaccine preparation, significantly enhancing the protective effect of the influenza vaccine, increasing the titer of influenza antigen-specific antibodies, and simultaneously increasing antibody levels for subtypes such as IgG1, IgG2a, IgG2b, and IgG3. In H1N1 challenge experiments, the influenza vaccine using 101-mer adjuvant achieved a 100% survival rate, with mice showing only a slight decrease in body weight followed by rapid recovery, and significantly reduced lung inflammation and pathological damage, indicating that the 101-mer adjuvant of this invention can provide complete protection after challenge and significantly reduce tissue damage. The antiviral activity of compound 101-mer is significantly superior to other compounds 18-mer, 19-mer, 27-mer, and LPS. Furthermore, compound 101-mer as an adjuvant did not exhibit observable toxicity and showed good tolerability even at high doses, demonstrating high safety, significantly better than LPS. Its mechanism of action is clear; it can act as a Dectin-2 agonist, activating the host immune system through the Syk / NF-κB signaling pathway. Attached Figure Description

[0014] Figure 1 The structural diagrams show the chemically synthesized mannocap arabinomannan compounds 101-mer (left), 27-mer (top right), 19-mer (middle right), and 18-mer (bottom right) derived from Mycobacterium tuberculosis.

[0015] Figure 2 The images show the phagocytosis of green fluorescent microspheres by mouse dendritic cell line DC2.4 promoted by 101-mer. The left image shows representative images of dendritic cells phagocytosing green fluorescent microspheres after stimulation with 18-mer, 19-mer, 27-mer, 101-mer, and LPS (scale bar: 100 μm). The right image is a quantitative statistical graph of fluorescence intensity corresponding to the left image. All data in the images are expressed as mean ± standard error (mean ± sem). Statistical analysis was performed using one-way ANOVA with n=3 replicates.

[0016] Figure 3The graph shows the effect of 101-mer as an adjuvant to enhance the immune protection of influenza vaccine; where ae represents the titer of HA-specific antibodies in serum as determined by ELISA: a is total IgG, b is IgG1, c is IgG2a, d is IgG2b, and e is IgG3; f is the result of the hemagglutination inhibition assay to determine the titer of functional antibodies; g is the curve of mouse body weight change after challenge; h is the survival curve of mice after challenge; i is the pathological result of H&E staining of lung tissue 8 days after challenge (day 36) (green arrows indicate the inflammatory cell infiltration area, scale bar = 100 μm).

[0017] Figure 4 The 101-mer spleen transcriptome sequencing and analysis are shown below; a) is a correlation heatmap; b) is a volcano plot of differentially expressed genes (DEGs), with the horizontal dashed line representing Padj=0.05, the left vertical dashed line representing the threshold of log2 (fold change) equal to -2, and the right vertical dashed line representing the threshold of log2 (fold change) equal to 2; c) is a heatmap of differentially expressed genes (DEGs); d) is the GO functional enrichment analysis of differentially expressed genes (MF: molecular function, CC: cellular component, BP: biological process); e) is the co-expression gene network of differentially expressed genes.

[0018] Figure 5 The diagrams show the molecular docking analysis of each total synthetic ManLAM compound (18-mer, 19-mer, 27-mer, and 101-mer) with Dectin-2; where a is the molecular docking diagram of 18-mer (fluorescent green backbone) with Dectin-2 CRD (gray); b is the molecular docking diagram of 19-mer (orange backbone) with Dectin-2 CRD (gray); c is the molecular docking diagram of 27-mer (purple backbone) with Dectin-2 CRD (gray); and d is the molecular docking diagram of 101-mer (blue backbone) with Dectin-2 CRD (gray).

[0019] Figure 6 The results of the safety evaluation of 101-mer and LPS are shown below; where a is the change in body weight of mice in each group; b is the tissue and organ coefficient of mice in each group; c is the serum biochemical index of mice in each group; d is the histological analysis of lung, liver, heart, spleen and kidney of mice in each group. The scale bar is 100 μm. Green arrows indicate inflammatory cell infiltration and yellow arrows indicate cytoplasmic sparseness. All data are expressed as mean ± standard error (sem). Statistical significance was tested by one-way ANOVA. n = 5 mice in each group.

[0020] Figure 7The safety evaluation results for 18-mer, 19-mer, and 27-mer mice are shown below; where a represents the change in body weight of mice in each group; b represents the tissue and organ coefficients of mice in each group; c represents the serum biochemical indicators of mice in each group; and d represents the histological analysis of the heart, liver, spleen, lung, and kidney of mice in each group. Scale bar = 100 μm. Data are expressed as mean ± standard error (sem). Statistical significance was determined by one-way ANOVA, with n = 5 mice in each group. Detailed Implementation

[0021] This invention provides the application of compound 101-mer in the preparation of influenza vaccines.

[0022] In this invention, compound 101-mer is preferably used as an adjuvant in influenza vaccines. Compound 101-mer is a fully synthetic mannose-capped arabinomannan (ManLAM) compound derived from Mycobacterium tuberculosis, possessing a high molecular weight glycosyl structure. The molecule contains a mannose cap and an arabinomannan backbone, and its structural formula is shown in Formula I. Ⅰ The compound 101-mer used in this invention was synthesized according to the preparation method described in the literature (Y. Ma, Y. Zhang, Y. Huang, Z. Chen, Q. Xian, R. Su, Q. Jiang, X. Wang, G. Xiao, J. Am. Chem. Soc. 2024, 146, 4112-4122.).

[0023] This invention utilizes compound 101-mer as an adjuvant in the preparation of influenza vaccines. Preferably, it enhances the immunoprotective effect of the influenza vaccine, increases the titer of total IgG antibodies specific to influenza antigens, and simultaneously increases the levels of IgG1, IgG2a, IgG2b, and IgG3 subtype antibodies. It provides complete protection after challenge, significantly reduces tissue damage, and exhibits antiviral activity significantly superior to other ManLAM compounds (18-mer, 19-mer, 27-mer) and LPS. Furthermore, compound 101-mer as an adjuvant has not shown observable toxicity and remains well-tolerated even at high doses, demonstrating high safety, significantly superior to LPS. The mechanism of action of compound 101-mer is clearly defined; preferably, it binds to the Dectin-2 receptor, acting as a Dectin-2 agonist to activate the host immune system through the Syk / NF-κB signaling pathway.

[0024] This invention provides the application of compound 101-mer in the preparation of influenza vaccine adjuvants.

[0025] In this invention, when compound 101-mer is used as an adjuvant for influenza vaccines, the preferred dosage in in vitro cell experiments is 1-50 μg / mL, more preferably 1 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL or 50 μg / mL. The recommended dosage in in vivo experiments in mice is preferably 10-50 μg / mouse, more preferably 10 μg / mouse, 20 μg / mouse, 30 μg / mouse, 40 μg / mouse or 50 μg / mouse.

[0026] The present invention also provides an influenza vaccine, wherein the influenza vaccine uses compound 101-mer as an adjuvant.

[0027] The preferred mass ratio of antigen to adjuvant in the influenza vaccine is (1:1) to (1:5), more preferably 1:1, 1:2, 1:3, 1:4 or 1:5. The preferred antigen is influenza virus hemagglutinin (HA).

[0028] In this invention, the influenza is preferably influenza A, and the influenza is preferably influenza caused by influenza A H1N1 virus.

[0029] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] Unless otherwise specified, the following embodiments are all conventional methods.

[0031] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0032] In the following examples, compounds 101-mer, 18-mer, 19-mer, and 27-mer were synthesized according to the preparation method described in the literature (Y.Ma, Y. Zhang, Y. Huang, Z. Chen, Q. Xian, R. Su, Q. Jiang, X. Wang, G. Xiao, J. Am. Chem. Soc. 2024, 146, 4112-4122.), and the structural formulas of each compound are shown in [reference needed]. Figure 1 .

[0033] Example 1 In vitro immune enhancement experiment Materials: Mouse dendritic cell line DC2.4 Culture medium: High glucose DMEM + 10% FBS + 100 U / mL penicillin + 100 μg / mL streptomycin Reagents: LPS, 18-mer, 19-mer, 27-mer and 101-mer (all at a concentration of 1 μg / mL), green fluorescent microspheres (2 μm, 1 μL / well) Methods: Healthy DC2.4 cells were cultured at a density of 1 × 10⁶ cells per well. 5 Cells were seeded at a density of 1 μg / mL in 12-well cell culture plates and cultured at 37°C and 5% CO2 for 12 hours. They were then divided into 5 groups (n=3): LPS, 18-mer, 19-mer, 27-mer, and 101-mer. The culture medium was discarded, and 1 mL of complete culture medium containing LPS (1 μg), 18-mer (1 μg), 19-mer (1 μg), 27-mer (1 μg), and 101-mer (1 μg) was added to each group, respectively. Incubation was continued for 12 hours. 1 μL of green fluorescent microspheres was added, and incubation continued for another 4 hours. The culture medium in the wells was discarded, and the cells were gently washed three times with pre-cooled phosphate-buffered saline (PBS). 100 μL of PBS was added to each well, and the fluorescence intensity of each well was immediately detected using a BioTek Cytation 1 fluorescence analysis system.

[0034] result: The phagocytic efficiency of DC2.4 cells on fluorescent microspheres was quantitatively analyzed by fluorescence intensity. The results are as follows: Figure 2 As shown, the compound 101-mer of the present invention significantly enhances the phagocytic ability of DC2.4 cells to green fluorescent microspheres, comparable to LPS; compared with 101-mer, 18-mer, 19-mer, and 27-mer have lower phagocytic enhancement effects.

[0035] Example 2 In vivo immune experiment in mice Materials: C57BL / 6 mice (6-8 weeks old), housed under SPF conditions. H1N1 influenza virus (amplified in chicken embryos and titrated using MDCK cells). Influenza virus hemagglutinin antigen, 18-mer, 19-mer, 27-mer, 101-mer, and LPS adjuvant. Enzyme-linked immunosorbent assay (ELISA) reagents: goat anti-mouse IgG (H+L) secondary antibody, and specific detection antibodies against IgG1, IgG2a, IgG2b, and IgG3 subtypes; 3,3′,5,5′-tetramethylbenzidine (TMB) substrate chromogenic solution. Hemagglutination inhibition assay (HAI) using 1% (v / v) chicken red blood cell suspension. Lung tissue pathology reagents: 4% paraformaldehyde, hematoxylin and eosin (H&E) staining reagent.

[0036] method: Experimental Groups: The following six groups were established, with 5 mice in each group. Ag group: HA antigen only (10 μg / time / mouse); LPS group: (10 μg HA + 10 μg LPS) / time / mouse; 18-mer group: (10 μg HA + 10 μg 18-mer) / time / mouse; 19-mer group: (10 μg HA + 10 μg 19-mer) / time / mouse; 27-mer group: (10 μg HA + 10 μg 27-mer) / time / mouse; 101-mer group: (10 μg HA + 10 μg 101-mer) / time / mouse.

[0037] Immunization schedule: Mice in each group were immunized twice intramuscularly with the above-described vaccine formulation on days 0 and 14. Ten days after the second immunization (day 24), blood was collected from the orbital sinus and serum was used to determine HA-specific antibody levels and HA-specific functional antibody titers. On day 28, 10×LD50 was administered... 50 Mice were challenged with a dose of H1N1 via the nasal cavity. From the date of challenge, mouse weight changes and survival status were monitored and recorded for 8 consecutive days. Mice whose weight decreased by more than 25% of their initial weight were considered to have reached the experimental endpoint and were humanely euthanized; this was included in the daily mortality rate. Eight days after challenge (day 36), all surviving mice were euthanized, and intact lung tissue was harvested, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with H&E. A semi-quantitative tissue damage score (0-4 points) was established based on indicators such as inflammatory cell infiltration and alveolar structural damage.

[0038] The level of HA-specific antibodies was determined by ELISA, which quantitatively detected total IgG and its subtypes (IgG1, IgG2a, IgG2b, IgG3). The specific steps were as follows: HA protein was diluted to 1 μg / mL with carbonate coating buffer and added to each well of a high-adsorption 96-well plate (100 μL). The plate was incubated overnight at 4°C. The coating solution was discarded, and PBS containing 2% BSA was added. The plate was blocked at 37°C for 2 hours. After washing three times with PBST containing 0.05% Tween-20, the plate was gently patted dry. The collected immunized mouse serum was serially diluted 1:2 with PBS containing 2% BSA and added to the wells. The plate was incubated at 37°C for 1 hour. After washing again, horseradish peroxidase-conjugated goat anti-mouse IgG (1:40000), IgG1 (1:10000), IgG2a (1:1000), IgG2b (1:1000), and IgG3 (1:1000) secondary antibodies were added to each well at 100 μL, and incubated at 37°C for 1 hour. After washing, 100 μL of TMB substrate solution was added to each well, and the reaction was incubated at room temperature in the dark for 10 minutes. Finally, 100 μL of 2M H2SO4 was added to terminate the reaction. The absorbance was measured at dual wavelengths of 450 nm and 630 nm using a microplate reader. The highest serum dilution corresponding to an absorbance 2.1 times higher than the average absorbance of negative serum was taken as the antibody endpoint titer.

[0039] The titer of HA-specific functional antibodies was detected by the HAI assay. The specific steps were as follows: Collected inactivated serum samples were serially diluted 1:2 and incubated with an equal volume of 4 hemagglutination units of H1N1 virus at room temperature. Subsequently, 1% chicken red blood cell suspension was added to each well and incubated at room temperature for 30 minutes. The HAI titer was defined as the reciprocal of the highest dilution of serum capable of completely inhibiting hemagglutination.

[0040] result: In vivo immunization experiments confirmed that when mice were co-immunized with influenza HA antigen as an adjuvant, the total IgG level induced by the 101-mer was comparable to that of the LPS adjuvant group, but significantly higher than that of the 18-mer, 19-mer, and 27-mer groups. Figure 3 (a) Further antibody subtype analysis showed that 101-mer broadly and evenly amplified the vaccine-induced IgG1, IgG2a, IgG2b, and IgG3 responses (a). Figure 3 The presence of the be in the hemagglutination inhibition assay indicates that it can induce a comprehensive immune response. Crucially, functional evaluation based on hemagglutination inhibition assays demonstrates that the 101-mer-induced antibody exhibits superior viral neutralization ability, with HAI titers superior to shorter ManLAM compounds (18-mer, 19-mer, and 27-mer) and the LPS adjuvant group (…). Figure 3(f) In the H1N1 virus challenge experiment, the 101-mer adjuvant vaccine provided 100% complete protection. Among all experimental groups, the 101-mer group mice experienced the smallest weight loss and the most rapid recovery. Figure 3 The survival rate (100%) of the g group was significantly higher than that of the 18-mer group (60%), 19-mer group (80%), 27-mer group (60%) and LPS group (80%). Figure 3 (h in the text). Further pathological examination of the lungs showed that the 101-mer adjuvant vaccine significantly reduced inflammatory cell infiltration and lung tissue damage after challenge; in contrast, all other groups showed moderate to severe pathological damage. Figure 3 (i in the text).

[0041] The above results demonstrate that the compound 101-mer of this invention can induce a high level and comprehensive specific humoral immune response, producing antibodies with high functionality (viral neutralization ability) and providing complete protection after viral challenge, significantly reducing lung pathological damage. Its adjuvant effect is significantly superior to traditional LPS adjuvants and 18-mer, 19-mer, and 27-mer, confirming its great potential as a highly effective vaccine adjuvant.

[0042] Example 3 Transcriptome analysis Spleen transcriptome sequencing methods: C57BL / 6 mice (6-8 weeks old), housed under SPF conditions, were randomly divided into two groups of 3 mice each: a 101-mer group (intramuscular injection of 10 μg 101-mer) and a control group (untreated). One week after administration, the mice were sacrificed, and the spleens were harvested and approximately 3 × 10⁻⁶ cells were obtained. 6 Immune cells were lysed, and high-quality total RNA was extracted for library construction. High-throughput sequencing was performed using the Illumina HiSeq platform, generating 150-bp paired-end reads. Data analysis included STAR genome alignment, DESeq2 differential analysis, BiNGO enrichment analysis, and WGCNA network analysis.

[0043] Transcriptomic results: Transcriptomic analysis identified 169 differentially expressed genes. The 101-mer treatment group showed a clear separation trend from the control group, indicating that 101-mer significantly altered the immunotranscriptional profile of the spleen. Figure 4 ac in the text). GO enrichment analysis showed that these differentially expressed genes were significantly enriched in immune-related biological processes, including immunoglobulin-mediated immune responses, B cell-mediated immunity, and antigen binding pathways. Figure 4(d) WGCNA network analysis further highlighted the key role of the Dectin-2 signaling pathway. Core gene nodes in this pathway, including Clec4n (Dectin-2 gene name), Syk, Card9, Bcl10, Malt1, Rela (p65), Nfkb1 (p105 / p50), Chuk (IKKα), Ikbkb (IKKβ), and Ikbke (IKKε), constitute a tightly interconnected co-expression network. Figure 4 (e). Dectin-2, a type C lectin receptor that recognizes mannose-rich glycans, is known to initiate the downstream Syk / NF-κB signaling cascade, promoting type I interferon production and thereby activating the host's innate immune system. This result supports the possibility that 101-mer adjuvants may enhance influenza vaccine efficacy through Dectin-2-mediated immune activation.

[0044] Example 4 Molecular docking Molecular docking method: The interaction modes and binding strengths between four ManLAM motifs (18-mer, 19-mer, 27-mer, 101-mer) and the mouse Decin-2 receptor (UniProt:Q9JKF4) were simulated and compared. The 3D structure of mouse Decin-2 predicted by AlphaFold (predicted TM-score = 0.71) was used, and spatial superposition with the human Decin-2 crystal structure (PDB:8ROV) was performed to verify the conservation of calcium ions at its sugar binding site, ensuring the reliability of the receptor model. Water molecules were removed, polar hydrogen was added, and Kollman charges were assigned using AutoDock Tools software. Ligand preparation: The carbohydrate ligands were first energy-minimized using the MM2 method in ChemBio 3D, followed by geometry optimization at ultra-high precision using the Forcite module to obtain the energy-optimal conformation. Docking process: Molecular docking calculations were performed using AutoDock Vina (v1.1.2). A lower binding free energy (ΔG, unit: kcal / mol) indicates a more stable binding.

[0045] Molecular docking results as follows Figure 5As shown in the ad, 18-mer, 19-mer, 27-mer, and 101-mer can all bind to the conserved calcium-dependent glycosyl-binding pocket of mouse Dectin-2, but their affinities differ significantly. 101-mer exhibits the lowest binding free energy (-14.4 kcal / mol) with Dectin-2, indicating the most stable binding. Its binding mechanism is stabilized by six hydrogen bonds (green, key residues include HIS-82, TRP-83, SER-85, GLU-120, TRP-164, and TRP-187) and extensive hydrophobic interactions. Figure 5 In contrast, docking of 18-mer (fluorescent green backbone) with Dectin-2 CRD (gray) showed moderate affinity (-11.7 kcal / mol), primarily through interactions via SER135 and TYR181 (green). Figure 5 (a) Docking of 19-mer (orange backbone) with Dectin-2 CRD (gray) showed similar affinity (-12.0 kcal / mol), and stable binding was achieved through an extended hydrogen bond network formed by SER133, ASN183, and GLU168 (green). Figure 5 (b) The docking of 27-mer (purple skeleton) with Dectin-2 CRD (gray) showed the weakest binding force (-11.1 kcal / mol), which was stabilized mainly by the interaction formed by GLU97 and GLU173 (green). Figure 5 (c in the text)

[0046] Example 5 Safety evaluation in mice Methods: C57BL / 6 mice (6-8 weeks old) were administered the following compound doses via tail vein injection, and divided into the following groups: 101-mer (10 μg), 101-mer (50 μg), 18-mer (50 μg), 19-mer (50 μg), 27-mer (50 μg), LPS (10 μg), LPS (50 μg), and a PBS control group (Control group 100 μL), with 5 mice in each group. Mouse weight was measured and recorded on days 0, 2, 4, 6, 8, 10, 12, and 14 post-injection. On day 14, mice were sacrificed under anesthesia, and heart, liver, spleen, lung, and kidney tissues were collected. The tissue-organ coefficient was calculated using the formula: Tissue-organ coefficient (%) = (Wet weight of tissue / organ / Body weight on the day) × 100%. Blood was collected from the orbital region of the mice, and serum was separated. The levels of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), urea (UREA), and creatinine (CREA) were measured using a fully automated animal blood biochemistry analyzer (BS-200, Mindray) to assess liver and kidney function. Collected heart, liver, spleen, lung, and kidney tissues were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with H&E. The tissue sections were observed, diagnosed, and imaged using an optical microscopy system (Leica).

[0047] Safety evaluation results indicate that the 101-mer exhibits good safety profile in vivo. For example... Figure 6 As shown, no significant weight loss was observed in mice treated with 101-mer (10 μg) and 101-mer (50 μg). Figure 6 a) Abnormal tissue and organ coefficients ( Figure 6 Abnormal fluctuations in serum biochemical indicators (including AST, ALT, ALP, UREA, CREA) or in the following categories: (b) Figure 6 (c). Histopathological analysis further confirmed that even at high doses, no obvious signs of inflammatory cell infiltration or necrosis were observed in multiple major organs, including the heart, liver, spleen, lungs, and kidneys, in the 101-mer treatment group mice. Figure 6 (d) It is noteworthy that, as a positive control, LPS adjuvant, even at lower doses, still induced significant lung and liver tissue damage ( Figure 6 In contrast, 101-mer remained well-tolerated at high doses. Similarly, no significant adverse reactions were observed with other ManLAM compounds (18-mer, 19-mer, and 27-mer). Figure 7 ).

[0048] In summary, the compound 101-mer of this invention retains strong adjuvant activity while successfully circumventing the serious toxicity issues associated with LPS, highlighting its significant advantages as a safe and clinically promising novel vaccine adjuvant. High doses of 101-mer were well tolerated, with no significant abnormalities in body weight, tissue / organ coefficients, or serum parameters, and no obvious inflammation or necrosis histologically. Low doses of LPS caused lung and liver damage, while the short-fragment compounds 18-mer, 19-mer, and 27-mer showed good safety.

[0049] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of compound 101-mer in the preparation of influenza vaccines.

2. The application according to claim 1, characterized in that, The compound 101-mer is used as an adjuvant in influenza vaccines.

3. The application according to claim 1 or 2, characterized in that, The compound 101-mer can enhance the immune protection effect of influenza vaccines.

4. The application according to claim 1 or 2, characterized in that, The compound 101-mer can act as a Dectin-2 agonist, activating the host immune system through the Syk / NF-κB signaling pathway.

5. Application of compound 101-mer in the preparation of influenza vaccine adjuvants.

6. An influenza vaccine, characterized in that, The influenza vaccine uses compound 101-mer as an adjuvant.

7. The influenza vaccine according to claim 6, characterized in that, The mass ratio of antigen to adjuvant in the influenza vaccine is (1:1) to (1:5).

8. The influenza vaccine according to claim 7, characterized in that, The antigen is influenza virus hemagglutinin.

9. The application according to any one of claims 1 to 5 or the influenza vaccine according to any one of claims 6 to 8, characterized in that, The influenza mentioned is influenza A.

10. The application according to any one of claims 1 to 5 or the influenza vaccine according to any one of claims 6 to 8, characterized in that, The influenza mentioned is caused by the H1N1 influenza virus.

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