Tp0136T cell epitope mRNA vaccine based on lipid nanoparticle delivery and application of Tp0136T cell epitope mRNA vaccine in syphilis prevention

Abstract

The invention relates to the technical field of mRNA vaccine research and development, and discloses a Tp0136T cell epitope mRNA vaccine based on lipid nanoparticle delivery and application of the Tp0136T cell epitope mRNA vaccine in syphilis prevention, the vaccine contains an mRNA sequence and an LNP delivery system, the mRNA sequence contains a Tp0136T1T cell epitope coding region (amino acid L477-S486, optimized by codon), a Cap 1 structure, optimized UTR and poly (A) tails of 65-76 adenosine, and psi or m5C is used for replacing trona; the LNP is prepared from SM-102, DSPC (Distearoyl Pyrrolidone), cholesterol and DMG-PEG (Dimethyl Glycol-Polyethylene Glycol) according to a molar ratio of 50 The particle size of the vaccine is less than or equal to 150nm, PDIlt; 0.2, the Zeta potential is-5 to-15 mV, and the encapsulation efficiency is greater than or equal to 93.47%. The preparation method comprises the steps of mRNA design synthesis, mRNA-LNP preparation characterization and in-vitro expression verification, through intramuscular injection inoculation, strong Th1 type and CD8 + CTL immune response can be induced, treponema pallidum load and skin ulcer rate can be reduced, and the method can be used for syphilis prevention.

Description

Technical Field

[0001] This invention belongs to the field of mRNA vaccine research and development technology, specifically relating to a Tp0136T cell epitope mRNA vaccine based on lipid nanoparticle delivery and its application in syphilis prevention. Background Technology

[0002] Syphilis is a chronic sexually transmitted infectious disease caused by Treponema pallidum. Although antibiotics such as penicillin are available for clinical treatment, the high recurrence rate, difficulty in identifying subclinical infections, and the increasing global spread mean that nearly ten million people worldwide are infected with the disease each year, with pregnant women and newborns facing particularly severe health threats. Currently, there is no approved syphilis vaccine, and vaccine development has long been stalled in the preclinical stage, becoming a critical challenge urgently needing a breakthrough in global public health.

[0003] Limitations of current syphilis vaccine development technologies: Current syphilis vaccine research largely focuses on B-cell epitopes and strategies for inducing antibody responses, such as candidate vaccines using Treponema pallidum membrane proteins like Tp0751 and Tp0326 as core antigens. However, studies have confirmed that Treponema pallidum is a bacterium with extremely low exposure to outer membrane antigens. Antibodies have limited effectiveness in clearing the pathogen, particularly in reducing the pathogen load at mucosal contact sites and failing to prevent early pathogen dissemination. Consequently, the protective efficacy of such vaccines falls far short of practical application requirements.

[0004] Further research has shown that T-cell-mediated delayed hypersensitivity (DTH) plays a central role in controlling Treponema pallidum replication, limiting the formation of skin and mucous membrane ulcers, and preventing systemic dissemination of the pathogen. Although some studies have attempted to design T-cell epitope vaccines using whole-protein or peptide forms, the resulting immune protection is still unsatisfactory due to limitations such as low antigen delivery efficiency, poor MHC molecule presentation efficiency, and insufficient induction of effector T cell activity, making it difficult to meet clinical prevention needs.

[0005] The antigen delivery platforms currently used in syphilis vaccine development have significant technical flaws, as detailed below:

[0006] Recombinant protein platforms: These platforms often use prokaryotic expression systems (such as E. coli) to express and purify fusion proteins like GST and PfTrx as antigen carriers. Their limitations include: the need for additional adjuvants to induce a strong immune response; difficulty in processing antigens within host cells and presenting them via the MHC-I pathway; and inability to effectively activate CD8. + T cells; poor antigenic structural stability and processability affect the effective exposure of T cell active epitopes.

[0007] Peptide vaccine platforms: Directly using synthetic peptides for immunization has problems such as poor stability, easy degradation by enzymes in the body, short half-life, limited retention time in the body, weak activation ability of T cells, and dependence on auxiliary carriers and adjuvants such as KLH to exert immune function. In practical applications, the immune effect fluctuates greatly.

[0008] Viral vector / adenovirus platform: Although it can activate cellular immunity to a certain extent, there are safety concerns—it may induce non-specific immune responses in the body and is prone to triggering viral vector-related immune escape, which limits its clinical translation potential.

[0009] In summary, current antigen delivery platforms have significant shortcomings in activating potent T-cell responses, achieving endogenous antigen expression, and controlling vaccine safety, and there is an urgent need to explore new delivery platforms to overcome these technological bottlenecks.

[0010] Advantages of mRNA Platforms and the Gap in Syphilis Vaccine Applications: In recent years, nucleotide-modified mRNA vaccine technology based on lipid nanoparticles (LNPs) has shown significant advantages, primarily in that it enables antigen self-expression within host cells, mimicking the natural infection process of pathogens; and it can effectively enter the MHC-I pathway, inducing potent CD8+ expression. + Cytotoxic T lymphocyte (CTL) response; rapid mRNA degradation in vivo with high safety; highly flexible platform that can quickly replace epitope coding sequences, suitable for vaccine development for a variety of pathogens; no need to add traditional adjuvants, avoiding adjuvant-related toxic side effects.

[0011] However, to date, no literature has reported the use of the mRNA-LNP vaccine platform for research on T-cell epitope vaccines related to syphilis prevention. This technological gap is a clear area that urgently needs to be filled.

[0012] Based on the limitations of the existing technology, this invention is the first to use an mRNA-LNP vaccine platform to deliver a specific T-cell epitope (Tp0136T1) derived from the Treponema pallidum Tp0136 protein, constructing an LNP vaccine encapsulating modified mRNA, and activating CD4 through in vivo expression. + and CD8 + T cells induce a significant Th1-type immune response, aiming to address the following key technical challenges: improving antigen expression efficiency and MHC-I pathway processing and presentation efficiency in host cells; and significantly enhancing CD8+. + It enhances the function of cytotoxic T cells, improves the ability to clear Treponema pallidum, effectively reduces RPR titer and pathogen load in animal models, and reduces the occurrence of skin and mucous membrane ulcers; avoids the problems of difficult antigen expression and purification and high adjuvant toxicity in traditional protein vaccines, and provides a feasible technical solution for the clinical translation of syphilis vaccines. Summary of the Invention

[0013] This invention provides a syphilis prevention vaccine based on an mRNA platform for delivering the Tp0136T cell epitope, along with its preparation and application methods. The core technology utilizes lipid nanoparticles (LNPs) to deliver chemically modified mRNA sequences, achieving efficient antigen expression within host cells and inducing a potent Th1-type immune response against the Tp0136T cell epitope and CD8+. + The cytotoxic immune response ultimately reduces the pathogenic load of Treponema pallidum and decreases the formation of mucosal ulcers, thus achieving the effect of syphilis prevention. At the same time, it solves the technical pain points of traditional vaccines, such as low delivery efficiency, insufficient immune activation, and poor safety.

[0014] To achieve the above objectives, the present invention provides the following technical solution:

[0015] A Tp0136T cell epitope mRNA vaccine based on lipid nanoparticle delivery, comprising an mRNA sequence and a lipid nanoparticle (LNP) delivery system;

[0016] The mRNA sequence comprises a Tp0136T1T cellular epitope coding region, a 5′ Cap1 structure, an optimized 5′ untranslated region (UTR), an optimized 3′ untranslated region (UTR), and a 3′ poly(A) tail; the amino acid sequence corresponding to the Tp0136T1T cellular epitope coding region is L477–S486, and this coding region has undergone codon optimization; the 3′ poly(A) tail contains 65–76 adenosines; the mRNA sequence uses modified nucleosides ψ or m5C to replace the natural bases;

[0017] The lipid nanoparticle (LNP) delivery system consists of SM-102, DSPC, cholesterol, and DMG-PEG in a molar ratio of 50:10:38.5:1.5.

[0018] Preferably, the mRNA vaccine has a particle size ≤150nm and a polydispersity index (PDI) <0.2 as determined by dynamic light scattering (DLS); a zeta potential of -5 to -15mV as determined by electrophoretic light scattering (ELS); an mRNA encapsulation efficiency ≥93.47% as determined by the RiboGreen method; a mRNA main peak integrity ≥89.6% as determined by capillary electrophoresis; and a mRNA cap efficiency ≥95.1% as determined by LC-MS.

[0019] Preferably, the length of the mRNA sequence was detected by capillary electrophoresis, and 89.6% of the transcripts in the transcript product were concentrated in the main peak of 213 nucleotides.

[0020] A method for preparing a Tp0136T cell epitope mRNA vaccine based on lipid nanoparticle delivery includes the following steps:

[0021] Step 1: mRNA Design and Synthesis

[0022] S1.1. Using a synthesis platform, design and synthesize mRNA containing the Tp0136T1T cell epitope coding region, 5′ Cap1 structure, optimized 5′ untranslated region (UTR), optimized 3′ untranslated region (UTR), and 3′ poly(A) tail. During the synthesis process, modified nucleosides ψ or m5C are used to replace the natural bases.

[0023] S1.2 Verify mRNA integrity by capillary electrophoresis, requiring the main peak ratio to be >89.6%; use LC-MS to detect the integrity of the mRNA's Cap structure and PolyA tail, requiring the Cap efficiency to reach 95.1%.

[0024] Step 2: Preparation and Physicochemical Characterization of mRNA-LNP

[0025] S2.1 The mRNA obtained in step one is encapsulated in an LNP composed of SM-102, DSPC, cholesterol and DMG-PEG in a molar ratio of 50:10:38.5:1.5 using a microfluidic system to form an mRNA-LNP vaccine;

[0026] S2.2. Dynamic light scattering was used to detect the particle size of the mRNA-LNP vaccine, requiring a particle size ≤150nm; polydispersity index (PDI) was detected, requiring PDI <0.2; zeta potential was detected by electrophoretic light scattering, requiring a zeta potential of -5 to -15mV; and the mRNA encapsulation efficiency was detected using the RiboGreen method, requiring an encapsulation efficiency ≥93.47%.

[0027] Step 3: In vitro expression validation

[0028] S3.1 Transfect HEK-293T cells with different doses of the LNP-mRNA obtained in step two;

[0029] S3.2. 48 hours after transfection, the expression of Tp0136T1 antigen was detected by immunofluorescence microscopy, confirming that the antigen expression was dose-dependent.

[0030] Preferably, the synthesis platform mentioned in step one is the synthesis platform of PerfectmRNACo. Ltd.

[0031] Preferably, the transfection dose of the mRNA-LNP vaccine in step three is 0–3 μg.

[0032] Application of a Tp0136T cell epitope mRNA vaccine delivered by lipid nanoparticles in syphilis prevention.

[0033] Preferably, the vaccine is administered via intramuscular injection; in animal models, for Balb / c mice, the dose is 5 μg, administered on Day 0, Day 21, and Day 42; for New Zealand white rabbits (NZW), the dose is 100 μg, administered on Day 0 and Day 21.

[0034] Preferably, the preventive effect of the vaccine is assessed by testing the following indicators in the vaccinated subjects:

[0035] (1) The DNA load of Treponema pallidum at the infection site of the inoculated animals was detected by qPCR.

[0036] (2) Detect the RPR titer and TPPA titer in the serum of the inoculated animals;

[0037] (3) Statistical analysis of the incidence and number of skin ulcers in infected animals;

[0038] The combined analysis of the above indicators was used to evaluate the protective effect of the vaccine. The number of ulcers was positively correlated with the RPR titer, and there was a significant correlation among Treponema pallidum DNA load, RPR titer, and number of ulcers.

[0039] As a preferred approach, a linear mixed-effects model was used to analyze the relationship between TPPA titer, RPR titer, post-challenge days, vaccine type, and ulcer number, with the LNP control group as the reference control group. When P < 0.05, it was considered that the factors among TPPA titer, RPR titer, post-challenge days, vaccine type, or ulcer number had a significant impact on the number of ulcers.

[0040] Compared with the prior art, the technical effects and advantages of the present invention are:

[0041] This invention constructs a highly efficient immune activation mechanism through the synergistic effect of a lipid nanoparticle (LNP) delivery system and functionalized mRNA. This system enables endogenous expression of antigens within host cells and completes antigen presentation via the MHC-I pathway, thereby simultaneously inducing potent Th1-type cellular immunity and CD8+. + Cytotoxic T lymphocyte (CTL) response—Th1-type cellular immunity is characterized by significantly elevated levels of IL-2 and IFN-γ, while CTL response is reflected in increased expression of Perforin and Granzyme B. This mechanism successfully addresses the key issue of weak cellular immune activation in traditional syphilis vaccines, providing core immune effect support for the body's clearance of Treponema pallidum.

[0042] Regarding its efficacy in preventing syphilis, this invention demonstrated significant advantages in a rabbit syphilis infection model. Experimental data showed that, compared to the 75% skin ulcer incidence rate in the blank LNP group, this vaccine reduced it to 16.7%; simultaneously, it significantly reduced the Treponema pallidum pathogen load at skin lesions, and the RPR titer in serum, reflecting the degree of tissue damage, also decreased significantly. This dual protective effect of reducing mucosal damage and clearing pathogens effectively reduces the risk of syphilis transmission through mucosal lesions, providing a practical and effective solution for syphilis prevention.

[0043] This invention safeguards these two key indicators through multiple design features. Regarding safety, the mRNA uses modified nucleosides (such as ψ and m5C) to replace natural bases, significantly reducing its inherent immunogenicity and avoiding non-specific inflammation. The DMG-PEG modification of LNP reduces the recognition of particles by opsonins in the blood and the clearance by phagocytes, decreasing its distribution in non-target tissues and further reducing non-specific immune responses. In terms of stability, the vaccine exhibits excellent physicochemical properties: PDI < 0.2, encapsulation efficiency ≥ 93.47%, and particle size ≤ 150 nm. Furthermore, it eliminates the need for traditional adjuvants (such as CFA), avoiding adjuvant-related toxicity and facilitating long-term storage and transportation.

[0044] This invention is the first to apply the mRNA-LNP platform to syphilis vaccine development, overcoming the limitation that traditional protein / peptide vaccines cannot effectively activate CD8. + The technical bottleneck of CTLs. By combining endogenous antigen expression with MHCI pathway presentation, this platform can directly activate the cytotoxic function of CTLs, specifically solving the core technical problem of "low exposure of outer membrane antigens and limited antibody clearance effect" of Treponema pallidum. This opens up a new technical path for syphilis vaccine development and fills the application gap of mRNA platforms in the field of syphilis prevention.

[0045] In terms of antigen selection, the specific T-cell epitope Tp0136T1 (L477–S486) of the Tp0136 protein was clearly identified, avoiding ineffective immunity and immunosuppression that may be caused by full-length proteins or mixed epitopes, ensuring that the immune response precisely targets the protective T-cell response. In terms of validation system, a complete process chain of "in vitro expression → mouse immune response → rabbit infection protection" was constructed, providing reproducible process parameters and quantitative evaluation standards, laying the foundation for clinical translation. In terms of industrialization, the preparation process (in vitro transcription, microfluidic encapsulation) is suitable for large-scale production, the key quality parameters meet GMP requirements, and the platform is highly flexible and can be extended to the development of vaccines for multiple pathogens, possessing good technology transfer value and market application prospects. Attached Figure Description

[0046] Figure 1 series (physicochemical properties and in vitro expression validation of mRNA-LNP vaccine)

[0047] Figure 1A The schematic diagram of the particle size distribution of the mRNA-LNP vaccine of this invention by dynamic light scattering (DLS) shows the hydration particle size range and concentration trend of the nanoparticles.

[0048] Figure 1B : A schematic diagram of the polydispersity index (PDI) of the mRNA-LNP vaccine of this invention by dynamic light scattering (DLS), reflecting the uniformity of particle size distribution;

[0049] Figure 1C : A schematic diagram of the zeta potential detected by electrophoretic light scattering (ELS) of the mRNA-LNP vaccine of this invention, demonstrating the colloidal stability of the vaccine system;

[0050] Figure 1D : A schematic diagram of the baseline fluorescence intensity of the mRNA-LNP vaccine of this invention for detecting encapsulation efficiency using the RiboGreen method (unbroken membrane state, detecting free mRNA);

[0051] Figure 1E : A schematic diagram of the RNA concentration standard curve for detecting encapsulation efficiency of the mRNA-LNP vaccine of this invention using the RiboGreen method, used for quantitative calculation of mRNA content;

[0052] Figure 1F : Schematic diagram of fluorescence intensity after membrane rupture for detecting encapsulation efficiency of the mRNA-LNP vaccine of this invention using the RiboGreen method (Triton X-100 membrane rupture, detection of total mRNA);

[0053] Figure 1G This is a schematic diagram of immunofluorescence detection of Tp0136T1 antigen after transfection of HEK-293T cells with the mRNA-LNP vaccine of this invention, showing the dose-dependent expression of antigen (including fluorescence comparison of LNP control group and different doses of mRNA-LNP group);

[0054] Figure 1H The left figure shows the appearance of the mRNA-LNP vaccine homogenate of the present invention (homogeneous transparent liquid); the right figure shows the SDS-PAGE electrophoresis diagram of the purified rPfTrx-Tp0136T1 fusion protein, verifying the protein purity (>90%).

[0055] Figure 1I : A schematic diagram of high performance liquid chromatography (HPLC) analysis of the Tp0136T1 synthesized polypeptide of the present invention, showing the retention time and purity peaks of the polypeptide;

[0056] Figure 1J This is a schematic diagram of mass spectrometry (MS) detection of the Tp0136T1 synthesized polypeptide of the present invention, showing the molecular ion peak of the polypeptide (e.g., [M+H)). +[M+2H] 2+ This verifies that the molecular weight is consistent with the theoretical value;

[0057] Figure 1K The schematic diagram of HPLC peak area percentage analysis of the Tp0136T1 synthesized polypeptide of this invention shows that the polypeptide purity reaches 99.709%.

[0058] Figure 2 series (Balb / c mouse immune response detection)

[0059] Figure 2A This invention presents a schematic diagram of the immunization and sample collection schedule for Balb / c mice, marking the immunization time points of Day 0, 21, and 42, as well as key milestones such as Day 56 euthanize (sacrifice) and spleen cell separation.

[0060] Figure 2B : Schematic diagram of ELISA detection results of anti-Tp0136T1IgG antibody titer in mouse serum after immunization (comparison of OD450 values ​​of each group, including blank control, LNP control, mRNA-LNP group, etc.);

[0061] Figure 2C : Schematic diagram of the detection results of IL-2 cytokine concentration in mouse spleen cells after stimulation with Tp0136T1 protein (comparison of pg / mL values ​​in each group, reflecting the Th1 immune response);

[0062] Figure 2D : Schematic diagram of the detection results of IFN-γ cytokine concentration in mouse spleen cells after stimulation with Tp0136T1 protein (comparison of pg / mL values ​​in each group, reflecting the Th1 immune response);

[0063] Figure 2E : Schematic diagram of the detection results of IL-4 cytokine concentration in mouse spleen cells after stimulation with Tp0136T1 protein (comparison of pg / mL values ​​in each group, reflecting the Th2 immune response);

[0064] Figure 2F : Schematic diagram of the detection results of IL-17 cytokine concentration in mouse spleen cells after stimulation with Tp0136T1 protein (comparison of pg / mL values ​​in each group, reflecting the Th17 immune response);

[0065] Figure 2G : Schematic diagram of the EdU incorporation experiment results of T cell proliferation in spleen cells after mouse immunization (comparison of the proportion of EdU-positive T cells in each group, reflecting the T cell activation capacity);

[0066] Figure 3 series (mouse T cell subsets and functional analysis)

[0067] Figure 3AThe present invention relates to CD45 in mouse spleen cells. + A schematic diagram of a flow cytometry cell sorting strategy (target cell populations are selected by labeling with SSC-A and CD45 antibodies);

[0068] Figure 3B The present invention relates to CD3 in mouse spleen cells. + Flow cytometry sorting strategy for T cells (in CD45) + CD3 cells were selected from the cell population. + T cells);

[0069] Figure 3C The present invention relates to mouse CD3 + CD4 in T cells + With CD8 + Flow cytometry sorting strategy for T cells (double-labeled antibodies distinguish T cell subsets);

[0070] Figure 3D The present invention relates to mouse CD4 + CD25 in T cells + Flow cytometry detection strategy for activation markers (Q5 / Q6 circle CD25) + cell);

[0071] Figure 3E The present invention relates to mouse CD4 + CD69 in T cells + Flow cytometry detection strategy for early activation markers (CD69 circled in Q1 / Q2) + cell);

[0072] Figure 3F This invention relates to mouse CD4 + CD62L in T cells + Flow cytometry detection strategy for initial T cell markers (Q9 / Q10 circle CD62L) + cell);

[0073] Figure 3G CD4 in mice of each group in this invention + CD25 in T cells + Schematic diagram of quantitative results of cell proportion (reflecting CD4) + (T cell activation level);

[0074] Figure 3H CD4 in mice of each group in this invention + CD62L in T cells + A schematic diagram of the quantitative results of cell proportions (reflecting the degree of conversion of naive T cells into effector T cells);

[0075] Figure 3ICD4 in mice of each group in this invention + CD69 in T cells + Schematic diagram of quantitative results of cell proportion (reflecting CD4) + (the degree of early T cell activation);

[0076] Figure 3J This invention relates to mouse CD4 + Schematic diagram of flow cytometry scatter plot of SSC-A and CD4 antibody-labeled T cells (CD4 circled) + T cells were used for subsequent intracellular factor detection.

[0077] Figure 3K This invention relates to mouse CD4 + IL-4 in T cells + Flow cytometry detection strategy for cells (Q2 circled IL-4) + Cells (reflecting Th2-type responses);

[0078] Figure 3L This invention relates to mouse CD4 + IFN-γ in T cells + Flow cytometry detection strategy for cells (Q6 circle IFN-γ) + Cells (reflecting Th1-type responses);

[0079] Figure 3M This invention relates to mouse CD4 + IL-17 in T cells + Flow cytometry detection strategy for cells (Q10 circle IL-17) + Cells (reflecting the Th17 type response);

[0080] Figure 3N This invention relates to mouse CD4 + A schematic diagram of flow cytometry detection of intracellular cytokines (IL-4, IFN-γ, IL-17) in T cells (to verify antibody specificity);

[0081] Figure 3O This invention relates to mouse CD8 + Schematic diagram of flow cytometry scatter plot of SSC-A and CD8 antibody-labeled T cells (CD8 circled) + T cells were used for subsequent molecular detection of cytotoxicity.

[0082] Figure 3P This invention relates to mouse CD8 + Granulase B in T cells + Flow cytometry detection strategy for cells (Q2 circle granzyme B) + Cells (reflecting CTL toxicity);

[0083] Figure 3Q This invention relates to mouse CD8 + Schematic diagram of flow cytometry detection strategy for perforin+ cells in T cells (Q2 circle perforin+ cells to reflect CTL toxicity);

[0084] Figure 3R This invention relates to mouse CD8 + A schematic diagram of the flow cytometry detection control of intracellular cytotoxic molecules (granzyme B, perforin) of T cells (to verify antibody specificity);

[0085] Figure 3S CD4 in mice of each group in this invention + IL-4 in T cells + A schematic diagram of the quantitative results of cell proportions (reflecting the strength of the Th2 immune response);

[0086] Figure 3T CD4 in mice of each group in this invention + IFN-γ in T cells + A schematic diagram of the quantitative results of cell proportions (reflecting the strength of the Th1 immune response);

[0087] Figure 3U CD4 in mice of each group in this invention + IL-17 in T cells + A schematic diagram of the quantitative results of cell proportions (reflecting the strength of the Th17 immune response);

[0088] Figure 3V CD8 in mice of each group in this invention + Granulase B in T cells + A schematic diagram of the quantitative results of cell proportion (reflecting the cytotoxic function of CTLs);

[0089] Figure 3W CD8 in mice of each group in this invention + A schematic diagram showing the quantitative results of the perforin+ cell ratio in T cells (reflecting the cytotoxic function of CTLs);

[0090] Figure 4 series (New Zealand white rabbit syphilis infection protection experiment)

[0091] Figure 4A This invention presents a schematic diagram of the immunization, Treponema pallidum challenge, and sample collection schedule for New Zealand white rabbits (NZW), with nodes marked such as Day 0 and 21 for immunization, Day 4 and 22 for challenge, and Day 6 and 3 for euthanize.

[0092] Figure 4B : Schematic diagram of ELISA detection results of anti-Tp0136T1IgG antibody titer in serum of rabbits after immunization (comparison of OD450 values ​​of each group);

[0093] Figure 4C This invention presents a schematic diagram of the qPCR detection results of Treponema pallidum DNA load in the skin lesions of rabbits infected with Treponema pallidum (comparison of gene copy numbers in each group).

[0094] Figure 4D This invention presents a schematic diagram of the detection results of TPPA titer in the serum of rabbits infected with Treponema pallidum (the changes in titer in each group verify the successful construction of the infection model).

[0095] Figure 4E This invention presents a schematic diagram of the detection results of RPR titer in the serum of rabbits infected with Treponema pallidum (comparison of titers in each group, reflecting the degree of tissue damage);

[0096] Figure 4F : Schematic diagram of statistical results of skin ulcer incidence, number of ulcers and diameter of lesions in each group of white rabbits after infection in this invention (including P value of each group, reflecting the protective effect of the vaccine);

[0097] Figure 4G : Schematic diagram of dark-field microscopy observation of skin lesions in rabbits at 7, 14 and 21 days after infection in each group of the present invention (comparing the ulcer healing status of each group);

[0098] Figure 4H Schematic diagram of the effect size (95% CI) of TPPA titer, RPR titer, and post-challenge days on ulcer number in the vaccine group (LNP-mRNA-Tp0136T1, polypeptide-Tp0136T1, rPfTrx-Tp0136T1) of this invention compared with the LNP control group.

[0099] Figure 4I A simplified analysis diagram of the effect size (95% CI) of each indicator (TPPA titer, RPR titer, and number of days after challenge) on the number of ulcers in the vaccine group compared with the LNP control group of this invention;

[0100] Figure 4J : A schematic diagram of the Spearman correlation analysis results of the number of ulcers and TPPA and RPR titers in infected rabbits after the invention (correlation coefficients rs and P values ​​are marked);

[0101] Figure 4K This invention presents a schematic diagram of the correlation analysis between the number of ulcers and TPPA / RPR titers based on RPR level stratification (distinguishing between low RPR group and high RPR group);

[0102] Figure 4L This invention presents a heatmap illustrating the correlation between skin lesion diameter, qPCR pathogen load, number of ulcers, TPPA titer, and RPR titer in infected rabbits (color intensity reflects the magnitude of the correlation coefficient).

[0103] Figure 5 The diagram shows the capillary electrophoresis detection of Tp0136T1 mRNA in this invention, which shows that 89.6% of the transcripts in the transcript product are concentrated in the main peak of 213 nucleotides, verifying the integrity of the mRNA.

[0104] Figure 6A This invention presents a schematic chromatogram of the LC-MS detection of the Cap structure (Cap1) of Tp0136T1 mRNA, showing the separation peak of the Cap structure;

[0105] Figure 6B This invention presents a schematic diagram of the mass spectrum of Tp0136T1 mRNA detected by LC-MS, showing the Cap structure ratio (Cap efficiency ≥ 95.1%) by using molecular ion peaks.

[0106] Figure 6C This invention presents a schematic chromatogram of the poly(A) tail of Tp0136T1 mRNA detected by LC-MS, illustrating the separation peak of the poly(A) tail;

[0107] Figure 6D The following is a schematic diagram of the LC-MS mass spectrum of Tp0136T1 mRNA, showing the poly(A) tail concentrated in 65–76 adenosines, verifying the structural integrity.

[0108] Figure 7 Key immune indicators in mice using the LNP-mRNA-Tp0136T1 vaccine of this invention (such as Edu positivity rate, CD4+, etc.) + IFN-γ + Proportion, CD8 + Granulase B + Spearman correlation analysis heatmap diagram between proportions, etc. (blue for positive correlation, red for negative correlation);

[0109] Figure 8 : A schematic diagram of Spearman correlation analysis heatmap of key immune indicators among mice in the rPfTrx-Tp0136T1 protein vaccine group of this invention (blue for positive correlation, red for negative correlation). Detailed Implementation

[0110] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0111] The present application will be further described in detail below with reference to the accompanying drawings;

[0112] A Tp0136T cell epitope mRNA vaccine based on lipid nanoparticle delivery includes an mRNA sequence and a lipid nanoparticle (LNP) delivery system; see Table 1 for the analysis of the functions of each component of the mRNA vaccine.

[0113] mRNA sequence

[0114] Antigen coding region: The core is the specific T cell epitope coding sequence (Tp0136T1) of the Tp0136 protein, corresponding to the amino acid fragment L477–S486. The coding region has been optimized by codon preference analysis and adapted to mammalian cell (such as HEK-293T cells, mouse / rabbit somatic cells) translation systems, which can improve antigen expression.

[0115] 5′ Regulatory structure: Contains a Cap1-type cap structure (7-methylguanosine), with a capping efficiency of ≥95.1% as detected by LC-MS. It can specifically bind to host cell translation initiation factors (such as eIF4E), improve the efficiency of mRNA translation initiation, and avoid direct degradation by intracellular exonucleases.

[0116] Untranslated regions (UTRs): Both the 5′ and 3′ ends employ optimized UTR sequences, which enhance the stability of mRNA in host cells, reduce degradation rate, and prolong the antigen expression window by regulating ribosome binding efficiency and mRNA secondary structure.

[0117] The 3′ poly(A) tail consists of 65–76 consecutive adenosines. Fragment length analysis has confirmed that the structure is intact and of uniform length. It can form a complex with intracellular poly(A) binding proteins (PABPs), which protects the 3′ end of mRNA from nuclease cleavage and promotes ribosome circulation, thereby improving translation efficiency.

[0118] Base modification: Modified nucleosides (such as pseudouridine ψ, 5-methylcytidine m5C) are used to replace some natural bases, reducing the inherent immunogenicity of mRNA (avoiding activation of TLR3 / 7 / 8 and other pattern recognition receptors to prevent non-specific inflammation) and improving chemical stability. As detected by capillary electrophoresis, the integrity of the main peak of the mRNA transcript is ≥89.6% (the main peak is concentrated in 213 nucleotides), which meets the quality standards of vaccine-grade mRNA.

[0119] Lipid nanoparticle (LNP) delivery system

[0120] The composition and formulation are as follows: It consists of SM-102 (cationic lipid, 50%), DSPC (saturated phospholipid, 10%), cholesterol (natural alcohol, 38.5%), and DMG-PEG (PEG-modified lipid, 1.5%) in molar proportions. Among them, SM-102 binds to mRNA through electrostatic interaction to achieve efficient encapsulation and assists mRNA in entering the cell via endocytosis; DSPC acts as a membrane "skeleton" to enhance structural rigidity and stability; cholesterol regulates membrane fluidity and promotes the fusion of LNP with the endosomal membrane to assist mRNA escape; DMG-PEG forms a "hydration layer" on the surface of LNP, reducing particle aggregation, prolonging the in vivo circulating half-life, and reducing the distribution in non-target tissues.

[0121] Key physicochemical properties: The mRNA-LNP vaccine formed by encapsulating mRNA with LNP, as characterized by physicochemical properties, has a particle size ≤150nm (Dynamic Light Scattering (DLS) detection), polydispersity index (PDI) <0.2 (DLS detection), zeta potential -5 to -15mV (electrophoretic light scattering (ELS) detection), and encapsulation efficiency ≥93.47% (RiboGreen method detection), ensuring that the vaccine is adapted to cell endocytosis, has good stability, and minimal mRNA loss.

[0122] Table 1. Analysis of the Functions of Each Component in mRNA Vaccine

[0123]

[0124]

[0125] This embodiment focuses on the preparation, characterization, in vitro validation, and in vivo efficacy evaluation of a Tp0136T cell epitope mRNA vaccine delivered by lipid nanoparticles (LNPs). Through specific experimental parameters and results, it fully presents the entire process of the vaccine from synthesis to application, and verifies its technical feasibility and syphilis prevention effect.

[0126] I. Preparation of Experimental Materials

[0127] (I) Key Reagents

[0128] Basic chemical reagents: modified nucleosides (pseudouridine ψ, 5-methylcytidine m5C, purchased from Sigma-Aldrich, catalog numbers U1128 and M6889 respectively); SM-102 cationic lipid (purchased from AvantiPolarLipids, catalog number 180005P); DSPC (distearylphosphatidylcholine, purchased from AvantiPolarLipids, catalog number 850365P); cholesterol (purchased from Sigma-Aldrich, catalog number C8667); DMG-PEG (1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol, purchased from AvantiPolarLipids, catalog number 880150P).

[0129] Detection kits and antibodies: RiboGreen kit (purchased from Thermo Fisher Scientific, catalog number R11490); rabbit anti-Tp0136T1 polyclonal antibody (in-house prepared, titer 1:5000); Alexa Fluor 488-labeled goat anti-rabbit secondary antibody (purchased from Invitrogen, catalog number A11034); flow cytometry antibodies (all purchased from BioLegend, USA, details see Table 2); CBA cytokine detection kit (purchased from BD, catalog number 551801).

[0130] Biological materials: HEK-293T cells (purchased from ATCC, catalog number CRL-3216); Balb / c mice (6-8 weeks old, female, purchased from Nanjing Model Animal Resource Bank, animal license number SCXK(Su)2022-0006); New Zealand white rabbits (NZW, 2-3 months old, female, purchased from the Comparative Medicine Center of Yangzhou University, animal license number SCXK(Su)2022-0008); Treponema pallidum strain Nichols (provided by the Microbiology Laboratory of the Dermatology Hospital of the Chinese Academy of Medical Sciences, catalog number TP-N-2023-001).

[0131] (II) Instruments and Equipment

[0132] Preparation instruments: microfluidic chip system (model NanoAssemblrIgnite, purchased from PrecisionNanosystems); ultrafiltration centrifuge tubes (molecular weight cutoff 100kDa, purchased from Millipore, catalog number UFC910096).

[0133] Characterization instruments: Dynamic light scattering system (DLS, model Zetasizer Nano ZS90, purchased from Malvern); Electrophoretic light scattering system (ELS, the same instrument as DLS); Capillary electrophoresis system (model Agilent 2100 Bioanalyzer, purchased from Agilent); LC-MS system (model Thermo Q Exactive, purchased from Thermo Fisher Scientific).

[0134] Detection instruments: Immunofluorescence microscope (Zeiss LSM880, purchased from Zeiss); qPCR instrument (ABI7500, purchased from Applied Biosystems); Flow cytometer (BDFACS CantoII, purchased from BDBiosciences).

[0135] (III) Preparation of Key Reagents

[0136] mRNA dissolution buffer: Citrate buffer (pH 4.0), weigh 2.101 g of citric acid and 2.941 g of sodium citrate, add ultrapure water to a final volume of 1000 mL, filter through a 0.22 μm filter membrane for sterilization, and store at 4 °C.

[0137] LNP organic phase: According to the molar ratio of SM102:DSPC:cholesterol:DMG-PEG = 50:10:38.5:1.5, accurately weigh each lipid component, dissolve it in anhydrous ethanol, and prepare a mixed solution with a concentration of 10 mmol / L. Prepare and use immediately.

[0138] Treponema pallidum bacterial culture: Treponema pallidum strain Nichols was inoculated into rabbit testicular tissue and cultured for 7 days. The bacterial culture was then collected and the concentration was adjusted to 1×10⁻⁶ with PBS. 6 Quantity / mL, aliquot and store at -80℃. Thaw and dilute to the required concentration before use.

[0139] Table 2. List of antibodies used in flow cytometry

[0140]

[0141]

[0142] II. mRNA Design and Synthesis

[0143] (I) mRNA structure design

[0144] Referring to the structural principles of mRNA in Figure 1, a Tp0136T1 mRNA sequence containing the following key elements was designed:

[0145] 5′ regulatory structure: Cap1-type cap structure (7-methylguanosine) ensures efficient translation initiation.

[0146] Untranslated regions (UTRs): Both the 5′ and 3′ ends use optimized UTR sequences (the 5′ UTR is derived from the human α-globin gene, and the 3′ UTR is derived from the human β-globin gene) to improve the stability of mRNA in mammalian cells.

[0147] Open reading frame (ORF): precisely encodes the Tp0136T1T cellular epitope (amino acid sequence L477–S486) and is optimized according to mammalian cell codon preferences (e.g., replacing rare codons with frequently used codons in mouse and rabbit cells).

[0148] 3′ poly(A) tail: Adding a poly(A) tail consisting of 65–76 consecutive adenosines prolongs the mRNA half-life.

[0149] (II) mRNA Synthesis and Quality Verification

[0150] Synthesis commissioned to PerfectmRNA Co. Ltd. for in vitro transcription technology to synthesize mRNA. Modified nucleosides (ψ, m5C) were used to replace the natural bases during the synthesis process. The reaction system included: template DNA (1 μg / μL), NTP mixture (containing modified nucleosides, 10 mmol / L), T7 RNA polymerase (20 U / μL), and RNase inhibitor (40 U / μL), and incubated at 37°C for 4 hours.

[0151] Integrity verification: via capillary electrophoresis ( Figure 5 mRNA integrity was detected using an Agilent 2100 Bioanalyzer with the RNA 6000 Pico Kit. The procedure was performed according to the kit instructions. The results showed that 89.6% of the transcripts were concentrated in the main peak at 213 nucleotides, which met the quality standards for vaccine-grade mRNA (80–95%).

[0152] Cap efficiency determination: The integrity of the cap structure was determined by LC-MS (ThermoQ Exactive). The chromatographic column was ACQUITYUPLCBEHC18 (2.1 mm × 100 mm, 1.7 μm). Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution. Gradient elution was used (0–10 min, 5–30% B; 10–15 min, 30–95% B) at a flow rate of 0.3 mL / min. The results showed that the cap efficiency reached 95.1%.

[0153] poly(A) tail verification: through fragment length analysis ( Figure 6C ,6D The poly(A) tail structure was confirmed using a 3′RACE kit (purchased from Takara, catalog number 6106), followed by amplification and sequencing verification. The results showed that the poly(A) tail length of the vast majority of transcripts was concentrated between 65 and 76 adenosines.

[0154] III. Preparation of mRNA-LNP Vaccine

[0155] (I) LNP Packaging Process

[0156] Phase preparation: The synthesized Tp0136T1 mRNA was dissolved in citrate buffer (pH 4.0) to prepare an aqueous phase with a concentration of 0.5 mg / mL; according to the above LNP group partition ratio, each lipid component was dissolved in anhydrous ethanol to prepare an organic phase.

[0157] Microfluidic mixing: The organic phase and aqueous phase were mixed at a volume ratio of 1:3 using a microfluidic device (model NanoAssemblrIgnite), with a total flow rate of 12 mL / min, a mixing channel temperature of 25 °C, and a reaction time of 5 min to form an mRNA-LNP complex.

[0158] Purification and concentration: The complex was purified using ultrafiltration centrifuge tubes (molecular weight cutoff 100kDa). The mixture was centrifuged at 3000×g for 15 min at 4℃ to remove unencapsulated mRNA and free lipids. This process was repeated 3 times. Finally, the vaccine was concentrated to an mRNA concentration of 1 mg / mL and stored at 4℃ for no more than 1 month.

[0159] (II) Physicochemical characterization of mRNA-LNP vaccines

[0160] See attached document Figures 1A to 1K The physicochemical properties of the prepared mRNA-LNP vaccine were analyzed using the detection method described above. The specific steps and results are as follows:

[0161] Particle size and PDI detection: 10 μL of vaccine sample was taken and diluted to 1 mL with PBS. Dynamic light scattering was performed using a Zetasizer Nano ZS90 instrument at a temperature of 25 °C and an equilibration time of 2 min. Each sample was tested 3 times. The results showed that the average particle size was 128.5 nm and the PDI was 0.156, which met the standards of ≤150 nm and <0.2 (Table 3).

[0162] Zeta potential detection: The diluted samples were taken and the zeta potential was detected using an electrophoretic light scattering instrument (same as DLS instrument). The detection cell was a disposable zeta cell. Each sample was detected 3 times. The results showed that the zeta potential was -9.8mV, which is in the stable range of -5 to -15mV (Table 3).

[0163] Encapsulation efficiency detection: The RiboGreen method was used. 20 μL of vaccine sample was divided into two groups: one group was directly added with RiboGreen reagent (1:1000 dilution) to detect free mRNA; the other group was added with 0.1% Triton X-100 to break the membrane before adding RiboGreen reagent to detect total mRNA. The fluorescence intensity was detected using an ELISA reader (TecanInfiniteM200) (excitation wavelength 485 nm, emission wavelength 520 nm). Encapsulation efficiency = (total mRNA fluorescence intensity - free mRNA fluorescence intensity) / total mRNA fluorescence intensity × 100%. The results showed that the encapsulation efficiency was 93.47% (Table 3).

[0164] Appearance observation: The vaccine appears as a homogeneous and transparent liquid with no sedimentation or layering, meeting the formulation requirements (Table 3).

[0165] Table 3 Physicochemical Characterization of mRNA-LNP Vaccine

[0166]

[0167] IV. In vitro expression verification experiment

[0168] (I) Cell Culture and Transfection

[0169] Cell preparation: HEK-293T cells were seeded in 24-well plates with coverslips and cultured in DMEM (containing 10% fetal bovine serum and 1% penicillin-streptomycin) at 37°C in a 5% CO2 incubator. Transfection was performed when the cells reached 70% confluence.

[0170] Grouping: Set up 5 groups, with 3 duplicate holes in each group. The specific processing is as follows:

[0171] Blank control group: Only Opti-MEM medium (0.5 mL / well) was added;

[0172] LNP control group: Transfected with blank LNP without mRNA (3 μg / well, diluted to 0.5 mL with Opti-MEM);

[0173] Low-dose mRNA-LNP group: Transfected with 1 μg LNP-mRNA-Tp0136T1 (diluted to 0.5 mL with Opti-MEM);

[0174] Medium-dose mRNA-LNP group: Transfected with 2 μg LNP-mRNA-Tp0136T1 (diluted to 0.5 mL with Opti-MEM);

[0175] High-dose mRNA-LNP group: Transfected with 3 μg LNP-mRNA-Tp0136T1 (diluted to 0.5 mL with Opti-MEM).

[0176] Transfection procedure: Using Lipofectamine 3000 reagent (Invitrogen, catalog number L3000015), follow the reagent instructions: add Lipofectamine 3000 (2 μL) and P3000 reagent (1 μL) to each well, incubate at room temperature for 5 min, then mix with the above-mentioned samples, add to a 24-well plate, and continue culturing for 48 h after transfection.

[0177] (II) Immunofluorescence detection

[0178] Sample preparation: Remove the coverslip from the 24-well plate and wash with PBS 3 times, 5 min each time; fix with 4% paraformaldehyde at room temperature for 15 min, and wash with PBS 3 times; permeate with 0.1% Triton X-100 at room temperature for 10 min, and wash with PBS 3 times; block with 5% BSA at room temperature for 30 min.

[0179] Antibody incubation: Add rabbit anti-Tp0136T1 polyclonal antibody (1:500 dilution, prepared with 5% BSA) and incubate overnight at 4°C; wash three times with PBS for 5 min each time the next day; add Alexa Fluor 488-labeled goat anti-rabbit secondary antibody (1:1000 dilution, prepared with 5% BSA) and incubate at room temperature for 1 h; wash three times with PBS, stain the nucleus with DAPI (5 μg / mL) for 5 min, and wash three times with PBS.

[0180] Results observation: Observation was performed using a fluorescence microscope (Zeiss LSM880) with excitation wavelengths of 488 nm (secondary antibody) and 350 nm (DAPI). Images were taken and fluorescence intensity was analyzed. Results are shown (see attached). Figure 1G No green fluorescence signal was detected in either the blank control group or the LNP control group. Specific fluorescence was observed in each dose group of mRNA-LNP, and the fluorescence intensity increased significantly with increasing mRNA dose, showing a dose-dependent effect, confirming that Tp0136T1 mRNA was successfully expressed in HEK-293T cells.

[0181] V. Mouse Immunization Experiment

[0182] (I) Experimental grouping and immunization procedure

[0183] Thirty 6-8 week old Balb / c mice were randomly divided into 5 groups of 6 mice each. The immunization regimens were as follows (see attached). Figure 2A The schedule, specific grouping and processing are as follows (Table 4):

[0184] Table 4. Grouping and Immunization Procedures for Mouse Immunization Experiments

[0185]

[0186]

[0187] (II) Sample Collection and Testing

[0188] Serum antibody detection (ELISA)

[0189] Sample collection: Blood was collected via the orbital cavity on Day 56, incubated at 37°C for 30 min, centrifuged at 4°C and 3000×g for 15 min, serum was separated and stored at -20°C.

[0190] ELISA Procedure: Coat the microplate with Tp0136T1 peptide (1 μg / mL, prepared with coating buffer pH 9.6), 100 μL / well, and incubate overnight at 4°C; the next day, wash three times with PBST (PBS containing 0.05% Tween-20), and block with 5% skim milk at room temperature for 1 h; serum is serially diluted with PBST (1:100, 1:1000, 1:10000, etc.), 100 μL / well, and incubated at 37°C for 1 h; PBST Wash three times, add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody (1:5000 dilution, catalog number A4416, Sigma-Aldrich) per well, and incubate at 37°C for 1 h; wash three times with PBST, add 100 μL of TMB chromogenic solution (catalog number T0440, Sigma-Aldrich) per well, and incubate at room temperature in the dark for 15 min; stop the reaction with 2 mol / L H2SO4, and measure the absorbance at 450 nm using a microplate reader.

[0191] Results Analysis (with appendix) Figure 2B The antibody titers in the mRNA-LNP group and the rPfTrx-Tp0136T1 protein group were significantly higher than those in the blank control group, LNP control group and peptide group (p<0.05). Among them, the antibody level in the rPfTrx-Tp0136T1 protein group was slightly higher, but the antibody in the mRNA-LNP group was more specific (no cross-reaction with irrelevant peptides).

[0192] splenic cell cytokine detection

[0193] Spleen cell preparation: Mice were sacrificed on Day 56, spleens were aseptically isolated, ground using a syringe plunger, and passed through a 70 μm cell sieve to obtain a single-cell suspension; erythrocyte lysis buffer (product number R7757, Sigma-Aldrich) was used for 5 min, followed by washing twice with PBS; the cell concentration was adjusted to 2 × 10⁶ cells / mL using RPMI-1640 medium containing 10% fetal bovine serum. 6 per mL.

[0194] Stimulation and detection: 1 mL of cell suspension was added to a 24-well plate, and Tp0136T1 protein (5 μg / mL) was added for stimulation. The plate was then incubated at 37°C and 5% CO2 for 72 h. The supernatant was collected, and the concentrations of IL-2, IFN-γ, IL-4 and IL-17 were detected using the CBA Cytokine Detection Kit (BD). The procedure was performed according to the kit instructions. The cells were then detected and analyzed by flow cytometry.

[0195] Results Analysis (with appendix) Figure 2C –F): Both the mRNA-LNP group and the rPfTrx-Tp0136T1 protein group showed typical Th1-type immune responses, with IL-2 (associated with) Figure 2C ) and IFN-γ (with Figure 2D The level was significantly higher than that of IL-4 (with appendix). Figure 2E ) and IL-17 (with Figure 2F Among them, the IL-2 level in the rPfTrx-Tp0136T1 group was slightly higher than that in the mRNA group (p<0.05), while only low levels of IL-4 were detected in the LNP control group.

[0196] T cell proliferation assay (EdU incorporation assay)

[0197] Experimental procedure: Take 2 × 103 spleen cells as described above. 6 Add EdU reagent (5 μmol / L, purchased from RiboBio, catalog number C10310-1) to cells, incubate at 37°C for 24 h; collect cells, wash twice with PBS, fix with 4% paraformaldehyde for 15 min, permeabilize with 0.5% Triton X-100 for 20 min; add Click reaction solution (Azide labeled with Alexa Fluor 594, catalog number C10310-1), react at room temperature in the dark for 30 min; stain nuclei with DAPI, and detect the proportion of EdU-positive T cells by flow cytometry.

[0198] Results Analysis (with appendix) Figure 2G The proportion of EdU-positive T cells in the mRNA-LNP group and the rPfTrx-Tp0136T1 protein group was significantly higher than that in the other three groups (p<0.05), confirming that both vaccines can effectively activate T cell proliferation.

[0199] (III) T cell subsets and functional analysis

[0200] Cell staining: 2 × 10⁻⁶ spleen cells were collected. 6Add surface antibodies (CD45, CD3, CD4, CD8, CD25, CD62L, CD69, diluted according to Table 1) to the cells (cells / mL), and incubate at 4°C for 30 min. Wash twice with PBS, add fixation and permeabilization agent (purchased from BD, catalog number 554714), and incubate at room temperature for 20 min. Add intracellular antibodies (IL-4, IFN-γ, IL-17, Perforin, Granzyme B, diluted according to Table 1), and incubate at 4°C for 30 min. Wash twice with PBS and analyze by flow cytometry.

[0201] Results Analysis

[0202] CD4 + T-cell activation (with) Figure 3G –I): mRNA-LNP group CD4 + CD25 in T cells + (Activation markers) and CD69 + The proportion of cells with early activation markers (CD62L) was significantly higher than that in the control group (p<0.05), and CD62L... + The proportion of CD4+ cells (initial T cell markers) was significantly reduced (p<0.05), indicating that CD4+ cells were significantly reduced. + T cells were effectively activated; intracellular cytokine detection showed (attached) Figure 3S –U), mRNA-LNP group CD4 + IFN-γ in T cells + The proportion of (Th1) cells was significantly increased, and IL-4 was elevated. + (Th2 type) and IL-17 + The low proportion of (Th17) cells confirms that Th1 cells are biased towards immune responses.

[0203] CD8 + T cell cytotoxicity (with appendix) Figure 3V –W): mRNA-LNP group CD8 + Granulase B in T cells + The proportion of perforin-rich cells was significantly higher than that of the blank control group, LNP control group, and peptide group (p<0.05), but there was no significant difference compared with the rPfTrx-Tp0136T1 protein group (p>0.05), indicating that the mRNA-LNP vaccine can effectively activate CD8. + Cytotoxic T cells (CTLs).

[0204] VI. Experiment on protection against Treponema pallidum infection in rabbits

[0205] (I) Experimental grouping and immunization procedure

[0206] Twelve 3-4 month old New Zealand White rabbits were randomly divided into four groups of three each. The immunization schedule was as follows (see attached). Figure 4AThe timetable, specific grouping and processing are as follows (Table 5):

[0207] Table 5. Rabbit Immunization Experiment Grouping and Immunization Procedure

[0208]

[0209] (II) Treponema pallidum challenge and sample detection

[0210] Challenge Experiment: Day 42, all rabbits had their backs shaved and were given subcutaneous injections at 8 points, each injection containing 1×10 5 A skin infection model was established using one Treponema pallidum strain (Nichols strain, diluted to 0.1 mL with PBS).

[0211] Serological testing

[0212] Sample collection: Blood samples were collected from the marginal ear vein before the challenge (Day 42) and 3 weeks after the challenge (Day 63). Serum was separated and stored at -20°C.

[0213] RPR assay: Toluidine red unheated serum assay kit (purchased from Shanghai Rongsheng Biotechnology, catalog number R1021) was used. The procedure was performed in accordance with the kit instructions. Results were expressed as titers (e.g., 1:1, 1:2, 1:4, etc.).

[0214] TPPA testing: The Treponema pallidum particle agglutination test kit (purchased from Fuji Rebio, catalog number TK730) was used. The procedure was performed in accordance with the kit instructions. Results are expressed as positive / negative.

[0215] Results Analysis (with appendix) Figure 4D –E): TPPA testing showed that all experimental groups were positive, confirming the successful construction of the infection model; RPR testing showed that the RPR titers of the mRNA-LNP group and the rPfTrx-Tp0136T1 protein group were significantly lower than those of the LNP control group and the peptide group (p<0.05), with the RPR titer of the mRNA-LNP group remaining at a low level of 1:4–1:8.

[0216] Skin ulcer observation and scoring

[0217] Observation method: Starting from the 7th day after the challenge, observe the ulcer formation at the injection site on the rabbit's back daily, record the number of ulcers and the maximum diameter (measured with calipers), and calculate the ulcer incidence rate (number of ulcers / total number of injection points × 100%).

[0218] Results Analysis (with appendix) Figure 4F–G): In the LNP control group, the ulcer incidence rate was 75% (6 / 8 injection sites), with an average ulcer diameter of 1.8 cm; in the polypeptide group, the ulcer incidence rate was 33.3% (2.67 / 8 injection sites), with an average ulcer diameter of 1.2 cm; in the mRNA-LNP group, the ulcer incidence rate decreased to 16.7% (1.33 / 8 injection sites), with an average ulcer diameter of 0.8 cm; the rPfTrx-Tp0136T1 protein group achieved complete protection, with an ulcer incidence rate of 0%.

[0219] Pathogen burden detection at skin lesion sites (qPCR)

[0220] Sample processing: On Day 63, rabbits were euthanized, and skin lesion tissue (approximately 0.5g) was collected from the back. The tissue was ground using a tissue homogenizer (QIAGENTissueLyserII). Total DNA was extracted using a DNA extraction kit (QIAGEN, catalog number 51304), and the DNA concentration was determined (using Nanodrop2000, ThermoFisherScientific).

[0221] qPCR procedure: The 16S rRNA gene of Treponema pallidum was used as the target gene, with primer sequences of: upstream 5′-GCTGGTGGTGACAAAGGTTG-3′, downstream 5′-TTGCGGGTGTGTAATGAAGG-3′; the internal control gene was rabbit GAPDH, with primer sequences of: upstream 5′-GGTGCTGAGTATGTCGTGGA-3′, downstream 5′-GGTGGTGAAGACGCCAGTA-3′. qPCR reaction system (20 μL): 2×SYBR GreenMix (10 μL, purchased from Takara, catalog number RR420A), forward and reverse primers (0.5 μL each, 10 μmol / L), DNA template (2 μL), enzyme-free water (7 μL); reaction program: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 5 s, 60℃ annealing and extension for 30 s, 40 cycles; melting curve analysis: 95℃ for 15 s, 60℃ for 1 min, 95℃ for 15 s.

[0222] Results Analysis (with appendix) Figure 4C The pathogen load of skin lesions in the mRNA-LNP group, rPfTrx-Tp0136T1 protein group and peptide group was significantly lower than that in the LNP control group (p<0.05). Among them, the pathogen load in the mRNA-LNP group and rPfTrx-Tp0136T1 protein group was the lowest (more than 80% lower than that in the control group).

[0223] (III) Correlation and Statistical Analysis

[0224] Correlation analysis: Spearman correlation analysis was used to explore the relationships between the indicators, and GraphPadPrism9 software was used for statistical analysis. The results show (see attached). Figure 4J –4L): The number of ulcers was significantly positively correlated with RPR titer (rs = 0.831, p < 0.05); there was a significant positive correlation among Treponema pallidum burden, RPR titer and number of ulcers at the lesion site (p < 0.05).

[0225] Linear mixed-effects model analysis: A linear mixed-effects model was used to analyze the relationship between TPPA titer, RPR titer, post-challenge days, vaccine type, and ulcer number. The LNP control group was used as a reference control group. Statistical analysis was performed using R software (lme4 package). The results showed (Table 6): RPR titer (estimated value 0.735, p = 0.023) and rPfTrx-Tp0136T1 vaccination (estimated value -1.675, p = 0.049) were significant factors affecting ulcer number. Although the mRNA-LNP vaccine did not reach statistical significance (p = 0.330), it still showed a trend of reducing ulcer number.

[0226] Table 6. Results of the linear mixed-effects model analysis (fixed effects)

[0227] Predictor variables Estimated value (95% confidence interval) Standard error t-value p-value TPPA titer 0.114(-0.738,0.967) 0.435 0.263 0.796 RPR titer 0.735(0.151,1.318) 0.298 2.467 0.023* Challenge the number of days -0.049(-0.412,0.314) 0.185 -0.266 0.793 LNP-mRNATp0136T1 -0.735(-2.111,0.641) 0.702 -1.047 0.330 Polypeptide-Tp0136T1 -1.322(-2.535,-0.110) 0.619 -2.138 0.070 rPfTrx-Tp0136T1 -1.675(-3.053,-0.298) 0.703 -2.385 0.049*

[0228] Note: The LNP control group was used as a reference control group. * indicates significance (P < 0.05).

[0229] VII. Verification of Alternative Solutions (Optional)

[0230] (I) PLGA-PEG Nanoparticle Delivery System

[0231] Preparation method: PLGA-PEG (purchased from Sigma-Aldrich, catalog number 719868) was dissolved in dichloromethane to prepare an organic phase with a concentration of 5 mg / mL; Tp0136T1 mRNA was dissolved in 1% PVA aqueous solution to prepare an aqueous phase; the organic phase and aqueous phase were mixed at a volume ratio of 1:5, ultrasonically emulsified (power 300W, time 3min), and stirred at room temperature for 2h to evaporate the solvent; ultrafiltration centrifugation (molecular weight cutoff 100kDa, 4℃, 3000×g) was performed 3 times to purify the mRNA to a concentration of 1 mg / mL.

[0232] Characterization and validation: The nanoparticles have an average particle size of approximately 180 nm, a PDI of <0.25, and an encapsulation efficiency of over 85%. In vitro transfection of HEK-293T cells showed dose-dependent expression of the Tp0136T1 antigen. Balb / c mouse immunization experiments (5 μg mRNA / mouse, intramuscular injection on Days 0, 21, and 42) showed that it could induce a Th1-biased immune response, with IL-2 and IFN-γ concentrations reaching 110-130 pg / mL and 170-190 pg / mL, respectively, after spleen cell stimulation.

[0233] (II) DNA Plasmid Expression System

[0234] Plasmid construction: A recombinant plasmid containing the Tp0136T1 coding sequence pcDNA3.1 was constructed (purchased from Invitrogen, catalog number V79020), and verified by enzyme digestion and sequencing.

[0235] Immunization and validation: In the New Zealand white rabbit model (100 μg plasmid / rabbit, immunized on Day 0 and 21, intramuscular injection combined with electroporation, 100V, 20ms pulse), the incidence of ulcers after challenge with Treponema pallidum was 41.7%, and the pathogen load of skin lesions was reduced by 75% compared with the empty plasmid control group.

[0236] VIII. Experimental Conclusions

[0237] This embodiment comprehensively validates the feasibility and protective effect of the "Tp0136T cell epitope mRNA vaccine based on lipid nanoparticle delivery" through mRNA design and synthesis, LNP encapsulation, in vitro expression verification, and animal immunization and infection protection experiments.

[0238] The successfully prepared mRNA-LNP vaccine has stable physicochemical properties, with an average particle size of 128.5 nm, a PDI of 0.156, a Zeta potential of -9.8 mV, and an encapsulation efficiency of 93.47%, meeting the standards for vaccine formulations.

[0239] In vitro experiments confirmed that the vaccine can efficiently express the Tp0136T1 antigen in HEK-293T cells, and the expression efficiency is dose-dependent.

[0240] Mouse immunization experiments showed that the vaccine could induce a strong Th1 immune response (increased IL-2 and IFN-γ) and CD8+. + Cytotoxic T cell activation (increased expression of granzyme B and perforin);

[0241] A rabbit model of Treponema pallidum infection showed that the vaccine significantly reduced the pathogen load at the site of skin lesions, reduced the incidence of skin ulcers (from 75% to 16.7%), and maintained a low level of RPR titer (1:4–1:8), demonstrating good syphilis prevention efficacy.

[0242] In summary, this embodiment provides complete experimental data support for the subsequent clinical translation, industrial scale-up, and patent application of the mRNA vaccine, confirming its application value in the field of syphilis prevention.

[0243] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A Tp0136 T-cell epitope mRNA vaccine based on lipid nanoparticle delivery, characterized in that, The mRNA sequence and a lipid nanoparticle (LNP) delivery system are included. The mRNA sequence comprises a Tp0136T1 T cell epitope coding region, a 5' end Cap1 structure, an optimized 5' untranslated region (UTR), an optimized 3' untranslated region (UTR), and a 3' end poly(A) tail; the Tp0136T1 T cell epitope coding region corresponds to the amino acid sequence L477-S486, and the coding region is codon-optimized; the 3' end poly(A) tail comprises 65-76 adenosines; the mRNA sequence uses modified nucleosides ψ or m5C to replace natural bases; The lipid nanoparticle (LNP) delivery system is composed of SM-102, DSPC, cholesterol, and DMG-PEG, and the molar ratio of the four is 50:10:38.5:1.

5.

2. A lipid nanoparticle-delivered Tp0136 T-cell epitope mRNA vaccine according to claim 1, characterized in that: The mRNA vaccine has a particle size of ≤150 nm and a polydispersity index (PDI) of <0.2 detected by dynamic light scattering (DLS); The Zeta potential is -5 to -15 mV detected by electrophoretic light scattering (ELS); the mRNA encapsulation rate is ≥93.47% detected by the RiboGreen method; The mRNA main peak integrity is ≥89.6% detected by capillary electrophoresis; The Cap efficiency of the mRNA is ≥95.1% detected by LC-MS.

3. The lipid nanoparticle-delivered Tp0136 T-cell epitope mRNA vaccine of claim 1, wherein: The length of the mRNA sequence is detected by capillary electrophoresis, and 89.6% of the transcripts in the transcription product are concentrated in the main peak of 213 nucleotides.

4. A method of manufacturing a lipid nanoparticle delivery based Tp0136 T cell epitope mRNA vaccine according to any one of claims 1 to 3, characterized in that, The steps include: Step one: mRNA design and synthesis S1.1, design and synthesize an mRNA comprising a Tp0136T1 T cell epitope coding region, a 5' end Cap1 structure, an optimized 5' untranslated region (UTR), an optimized 3' untranslated region (UTR), and a 3' end poly(A) tail using a synthesis platform, and use modified nucleosides ψ or m5C to replace natural bases during synthesis; S1.2, verify the mRNA integrity by capillary electrophoresis, requiring the main peak proportion to be >89.6%; detect the Cap structure and PolyA tail integrity of the mRNA by LC-MS, requiring the Cap efficiency to reach 95.1%; Step two: preparation and physical and chemical characterization of mRNA-LNP S2.1, encapsulate the mRNA obtained in step one in the LNP composed of SM-102, DSPC, cholesterol, and DMG-PEG with a molar ratio of 50:10:38.5:1.5 through a microfluidic system to form an mRNA-LNP vaccine; S2.2, detect the particle size of the mRNA-LNP vaccine using dynamic light scattering, requiring the particle size to be ≤150 nm; detect the polydispersity index (PDI), requiring the PDI to be <0.2; detect the Zeta potential by electrophoretic light scattering, requiring the Zeta potential to be -5 to -15 mV; detect the mRNA encapsulation rate by the RiboGreen method, requiring the encapsulation rate to be ≥93.47%; Step three: in vitro expression verification S3.1, transfect HEK-293T cells with different doses of LNP-mRNA obtained in step two; S3.2, 48 hours after transfection, the expression of Tp0136 T1 antigen was detected by immunofluorescence microscopy, and it was confirmed that the antigen expression was dose-dependent.

5. A method of preparing a lipid nanoparticle-delivered Tp0136 T-cell epitope mRNA vaccine according to claim 4, characterized by: The synthetic platform in step one is the synthetic platform of Perfect mRNA Co. Ltd.

6. A method of preparing a lipid nanoparticle-delivered Tp0136 T-cell epitope mRNA vaccine according to claim 4, characterized by: The transfection dose of mRNA-LNP vaccine in step three is 0-3 μg.

7. The lipid nanoparticle delivery-based Tp0136 T cell epitope mRNA vaccine of any one of claims 1-3 for use in the prevention of syphilis.

8. Use of a lipid nanoparticle-delivered Tp0136 T-cell epitope mRNA vaccine according to claim 7 for the prevention of syphilis, characterized in that: The vaccine is inoculated by intramuscular injection; in animal models, the inoculation dose for Balb / c mice is 5 μg, and the inoculation time points are Day 0, Day 21 and Day 42; for New Zealand white rabbits (NZW), the inoculation dose is 100 μg, and the inoculation time points are Day 0 and Day 21.

9. Use of a lipid nanoparticle-delivered Tp0136 T-cell epitope mRNA vaccine according to claim 7 for the prevention of syphilis, characterized in that: The preventive effect of the vaccine is evaluated by detecting the following indicators of the inoculation object: (1) The syphilis spirochete DNA load in the infected site of the inoculated animal is detected by qPCR technology; (2) The RPR titer and TPPA titer in the serum of the inoculated animal are detected; (3) The incidence of skin ulcers and the number of ulcers after infection of the inoculated animal are counted; Through the joint analysis of the above indicators, the protective effect of the vaccine is evaluated, wherein the number of ulcers is positively correlated with the RPR titer, and there is a significant correlation between the syphilis spirochete DNA load, the RPR titer and the number of ulcers.

10. Use of a lipid nanoparticle-delivered Tp0136 T-cell epitope mRNA vaccine according to claim 9 for the prevention of syphilis, characterized in that: The relationship between the TPPA titer, the RPR titer, the number of days after challenge, the type of vaccine inoculation and the number of ulcers is analyzed by linear mixed effect model, and the LNP control group is used as the reference control group; when P<0.05, it is considered that the factors in the TPPA titer, the RPR titer, the number of days after challenge, the type of vaccine inoculation or the number of ulcers have a significant effect on the number of ulcers.

Images