An intramuscularly delivered tp0326 recombinant adenovirus syphilis vaccine

Abstract

The application discloses an intramuscular delivery Tp0326 recombinant adenovirus syphilis vaccine, which comprises a recombinant adenovirus rAd5-Tp0326; the recombinant adenovirus rAd5-Tp0326 is a carrier of a replication-defective human adenovirus type 5, a Tp0326 full-length gene of Treponema pallidum is inserted into an adenovirus expression vector pshuttle-IRES-rGFP-1, and the adenovirus expression vector pshuttle-IRES-rGFP-1 is constructed by homologous recombination with an adenovirus backbone plasmid pAdEasy-1; the application adopts the replication-defective adenovirus type 5 as the carrier, has high safety, is non-pathogenic, can efficiently accommodate the Tp0326 full-length gene, supports eukaryotic expression of the antigen, makes the conformation of the expressed Tp0326 antigen closer to a natural state, can induce specific antibodies against natural epitopes, and significantly improves immunogenicity; the vaccine is delivered by an intramuscular injection mode, the operation is simple, the delivery efficiency is high, the vaccine can quickly induce the body to produce humoral and cellular immune responses, effectively reduces development of pathological changes after infection, and reduces bacterial load; the specific antibodies induced by the vaccine have strong anti-adhesion and opsonophagocytic activities.

Description

Technical Field

[0001] This invention relates to the field of vaccine technology, specifically to an intramuscularly delivered Tp0326 recombinant adenovirus syphilis vaccine. Background Technology

[0002] Syphilis is a sexually transmitted and vertically transmitted infectious disease caused by Treponema pallidum subsp. pallidum. Given the enormous disability-adjusted life-year (DALY) burden of syphilis, the high incidence of serious adverse pregnancy outcomes, and the severe clinical consequences of congenital syphilis associated with vertical transmission of Treponema pallidum, the development of a preventative vaccine is a critical public health priority for ensuring the health of future generations.

[0003] The outer membrane protein Tp0326 (also known as BamA protein) of *Treponema pallidum* is highly conserved. Its extracellular loop contains an immunodominant epitope that can induce antibodies with opsonization activity, making it an important candidate antigen for syphilis vaccines. However, there are limitations to the expression systems used for syphilis vaccines. Firstly, prokaryotic expression systems cannot achieve eukaryotic post-translational modification of the Tp0326 antigen, and DNA vaccines rely on the host cell's own expression mechanism. Neither can efficiently support stable expression of the full-length Tp0326 gene, and they cannot effectively mimic the expression environment of the Tp0326 antigen in natural *Treponema pallidum*. This results in a significant difference in the conformation of the expressed Tp0326 antigen compared to its natural state, and the surface-exposed antigenic epitope of its β-barrel domain cannot be properly presented, failing to effectively induce the body to produce specific antibodies against the natural epitope. Secondly, there is insufficient immunogenicity. The antigen conformation deviates from the natural state, and the antigen expressed in prokaryotes lacks natural modification. The cellular immune response induced by DNA vaccines is also relatively weak, resulting in low overall immunogenicity of the vaccine. It is difficult to stimulate the body to produce sufficient titers of functional antibodies, and thus cannot fully exert its preventive effect against Treponema pallidum infection. Thirdly, some existing technologies use attenuated live vaccine vectors, which have the potential risk of virulence recovery, and safety cannot be fully guaranteed. While prokaryotes expressed subunit vaccines have higher safety, they further limit the immunogenicity of the antigen, and cannot meet the dual requirements of safety and immune efficacy.

[0004] To this end, we have launched an intramuscularly delivered Tp0326 recombinant adenovirus syphilis vaccine. Summary of the Invention

[0005] The purpose of this invention is to provide an intramuscularly delivered Tp0326 recombinant adenovirus syphilis vaccine to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: an intramuscularly delivered Tp0326 recombinant adenovirus syphilis vaccine, comprising: recombinant adenovirus rAd5-Tp0326; wherein the recombinant adenovirus rAd5-Tp0326 is constructed by inserting the full-length gene of Treponema pallidum Tp0326 into the adenovirus expression vector pshuttle-IRES-rGFP-1 and homologously recombinating it with the adenovirus backbone plasmid pAdEasy-1; The recombinant adenovirus rAd5-Tp0326 can express the Tp0326 antigen in host cells. The vaccine is delivered via intramuscular injection to induce a specific immune response against Treponema pallidum and inhibit Treponema pallidum infection and transmission.

[0007] Preferably, the *Treponema pallidum* Tp0326 gene is the full-length coding sequence of *Treponema pallidum* strain *Nichols*, and the expressed Tp0326 antigen is a recombinant protein containing an N-terminal 6×His tag.

[0008] Preferably, the method for constructing the recombinant adenovirus rAd5-Tp0326 includes the following steps: 1. The full-length Tp0326 gene was amplified from the genomic DNA of Treponema pallidum by PCR; 2. The amplified Tp0326 gene was cloned into the pshuttle-IRES-rGFP-1 shuttle plasmid through restriction enzyme sites to construct a recombinant shuttle plasmid; 3. The recombinant shuttle plasmid was linearized with restriction endonuclease PmeI and co-transformed with the pAdEasy-1 backbone plasmid into E. coli BJ5183 competent cells for homologous recombination. 4. Screen for positive recombinant adenovirus plasmids, linearize them with restriction endonuclease PacI, transfect HEK293T cells with Lipofectamine®LTX, and package them to obtain recombinant adenovirus rAd5-Tp0326; 5. The recombinant adenovirus rAd5-Tp0326 was amplified in HEK293T cells and purified by CsCl density gradient centrifugation to obtain high-purity recombinant adenovirus particles.

[0009] Preferably, the purified recombinant adenovirus rAd5-Tp0326 titer in step 5 is 1×10⁻⁶. 10 VP / mL, the virus particles have a complete icosahedral morphology.

[0010] Preferably, the intramuscular delivery dose of the vaccine is: for New Zealand white rabbits, a single intramuscular injection of 1 mL, containing 1×10⁻⁶ mmol / L. 8PFU recombinant adenovirus rAd5-Tp0326; for BALB / c mice, a single intramuscular injection of 50 μL containing 1×10 7 PFU recombinant adenovirus rAd5-Tp0326.

[0011] Preferably, the immunization schedule for the vaccine consists of two intramuscular injections, with a 21-day interval between the initial immunization and the booster immunization, and the same dosage for both immunizations.

[0012] Preferably, the specific immune response induced by the vaccine includes: generating high-titer anti-Tp0326 IgG antibodies, inducing a Th2-biased immune response (IgG1 / IgG2a>1), promoting T lymphocyte antigen-specific proliferation, and increasing IL-17a cytokine levels.

[0013] Preferably, the specific antibodies induced by the vaccine can inhibit the adhesion of Treponema pallidum to cottontail rabbit epithelial Sf1Ep cells and promote the opsonization and phagocytosis of Treponema pallidum strains Nichols and SS14 by mouse bone marrow-derived macrophages or rabbit peritoneal macrophages.

[0014] Preferably, the vaccine is used to prevent infection with Treponema pallidum, which can reduce the development of skin lesions after infection, reduce the load of Treponema pallidum in the lesion tissue, and block the transmission of Treponema pallidum between hosts.

[0015] Preferably, after the recombinant adenovirus rAd5-Tp0326 infects HEK293T cells and Sf1Ep cells, it can effectively express the Tp0326 antigen, and the expressed antigen is verified by Western blot to show a specific band at the expected molecular weight.

[0016] Compared with the prior art, the beneficial effects of the present invention are: (1) By using a replication-defective type 5 adenovirus as a vector, it is safe, non-pathogenic, and can efficiently accommodate the full-length Tp0326 gene, supporting the eukaryotic expression of the antigen, making the expressed Tp0326 antigen conformation closer to the natural state, and can induce the production of specific antibodies against the natural epitope, significantly improving immunogenicity. (2) The intramuscular injection method is simple to operate and has high delivery efficiency. It can quickly induce humoral and cellular immune responses in the body. Moreover, the protective effect of intramuscular delivery is better than that of other methods such as intranasal delivery. It can effectively reduce the development of lesions after infection and reduce bacterial load. (3) The specific antibodies induced by the vaccine have strong anti-adhesion and opsonization activities. They can not only inhibit the infection of host cells by Treponema pallidum, but also promote the clearance of pathogens by macrophages and block the spread of Treponema pallidum. Attached Figure Description

[0017] Figure 1 This is a schematic block diagram of the recombinant adenovirus syphilis vaccine of the present invention; Figure 2 This is a schematic diagram illustrating the preparation of the P0326 recombinant defective adenovirus of the present invention; Figure 3 This is a schematic diagram illustrating the differential activation and functional polarization of T cell subsets in mice after vaccination with the TP0326 vaccine according to the present invention. Figure 4 This is a schematic diagram illustrating the in vitro phagocytic activity of serum antibodies from mice immunized with the vaccine of this invention against Nichols reference strain and clinical SS14L strain. Figure 5 This is a schematic diagram illustrating the in vitro phagocytic activity of rabbit serum antibodies immunized with the vaccine of this invention against Nichols reference strain and clinical SS14L strain. Figure 6 This is a schematic diagram illustrating the characteristics of the humoral and cellular immune responses induced by the vaccine before pathogen challenge according to the present invention. Figure 7 This invention relates to the subcutaneous region of the rabbit's back after vaccination and subsequent administration of a high dose of Treponema pallidum (1×10⁻⁶ per site). 5 A schematic diagram of the pathological phenotypic changes that occur after a challenge (individual bacteria); Figure 8 This is a schematic diagram showing the herpes lesions on the back skin of rabbits of different immune groups and the results of dark-field examination. Figure 9 This invention relates to the subcutaneous region of the rabbit's back after vaccination and subsequent administration of a low dose of Treponema pallidum (1×10⁻⁶ per site). 2 A schematic diagram of the pathological phenotypic changes that occur after a challenge (individual bacteria). Detailed Implementation

[0018] 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.

[0019] Example 1

[0020] Please see Figure 1-9 The present invention provides a technical solution: an intramuscularly delivered Tp0326 recombinant adenovirus syphilis vaccine, the construction and purification of recombinant adenovirus rAd5-Tp0326; 1. Materials Preparation: HEK293T cells were purchased from ATCC (catalog number CRL-11268). pshuttle-IRES-rGFP-1 shuttle plasmid, pAdEasy-1 backbone plasmid, and Escherichia coli BJ5183 competent cells were all routinely preserved. Restriction endonucleases PmeI, PacI, PCR primers, and Lipofectamine®LTX transfection reagent were all purchased from routine commercial channels. The Nichols strain of Treponema pallidum was preserved by the Dermatology Hospital of Southern Medical University.

[0021] 2. PCR amplification of the Tp0326 gene: The full-length Tp0326 gene was amplified from the genomic DNA of *Treponema pallidum* strain Nichols using specific primers. The PCR reaction conditions were: 95℃ pre-denaturation for 5 minutes, 95℃ denaturation for 30 seconds, 58℃ annealing for 30 seconds, 72℃ extension for 2 minutes, for a total of 35 cycles, and a final extension at 72℃ for 10 minutes. The amplified product was verified by agarose gel electrophoresis, and the target fragment was recovered.

[0022] 3. Construction of recombinant shuttle plasmid: The recovered Tp0326 gene was cloned into the pshuttle-IRES-rGFP-1 shuttle plasmid through restriction enzyme sites to construct the recombinant shuttle plasmid pshuttle-Tp0326. The recombinant plasmid was transformed into Escherichia coli BJ5183 competent cells, positive clones were screened, and the sequence was confirmed to be correct by DNA sequencing.

[0023] 4. Construction of recombinant adenovirus plasmid: The correctly sequenced recombinant shuttle plasmid pshuttle-Tp0326 was linearized with PmeI and co-transformed with the pAdEasy-1 backbone plasmid into E. coli BJ5183 competent cells. After incubation at 37°C for 18 hours, single colonies were picked, plasmids were extracted, and homologous recombination was confirmed by enzyme digestion and DNA sequencing to obtain the recombinant adenovirus plasmid pAd-Tp0326.

[0024] 5. Packaging and Amplification of Recombinant Adenovirus: pAd-Tp0326 was linearized with PacI, recovered by agarose gel, and transfected into HEK293T cells using Lipofectamine® LTX. The cells were incubated at 37°C for 6-7 days in a 5% CO2 incubator. After observing GFP fluorescence and cytopathic effects, the cells and culture supernatant were collected. The cells were subjected to three freeze-thaw cycles, and the viral supernatant was collected by centrifugation. This was the stock solution of recombinant adenovirus rAd5-Tp0326. The stock solution was continuously amplified in HEK293T cells to obtain a large amount of recombinant adenovirus.

[0025] 6. Purification and Identification of Recombinant Adenovirus: The amplified recombinant adenovirus was purified by CsCl density gradient centrifugation. The viral band was collected, and CsCl was removed by dialysis to obtain purified rAd5-Tp0326 viral particles. The viral morphology was observed by transmission electron microscopy, confirming that the viral particles had a complete icosahedral structure. The viral titer was determined by TCID50, and the result showed that the viral titer was 1×10⁻⁶. 10 VP / mL; Western blot was used to verify the expression of the Tp0326 antigen, and the results showed a specific band at the expected molecular weight, proving that the recombinant adenovirus was successfully constructed.

[0026] Example 2

[0027] Immunogenicity testing of intramuscularly delivered Tp0326 recombinant adenovirus syphilis vaccine 1. Animal grouping and immunization: 6-8 week old female BALB / c mice were randomly divided into 4 groups of 5 mice each: rAd5-Tp0326 intramuscular immunization group, Ad5 (wt) intramuscular immunization group, rTp0326 protein immunization group, and saline control group; rAd5-Tp0326 intramuscular immunization group: 50 μL containing 1×10⁻⁶ protein was injected intramuscularly on day 0 and day 21. 7 PFUrAd5-Tp0326 in normal saline; Ad5 (wt) intramuscular immunization group: 50 μL containing 1×10 on day 0 and day 21. 7 PFUAd5 (wt) in physiological saline; rTp0326 protein immunization group: 200 μL of complete Freund's adjuvant emulsion containing 50 μg g Tp0326 protein was injected intraperitoneally on day 0, and 200 μL of incomplete Freund's adjuvant emulsion containing 25 μg g Tp0326 protein was injected intraperitoneally on day 21; control group: 50 μL of physiological saline was injected intramuscularly on day 0 and day 21.

[0028] 2. Serum antibody detection: Blood was collected via tail vein on days 21 and 42, and serum was separated. The titer of anti-Tp0326 IgG antibody was detected by ELISA. The results showed that the antibody titer of the intramuscular rAd5-Tp0326 immunization group was ≥50,000 on day 42, which was significantly higher than that of the Ad5 (wt) immunization group and the control group (P<0.05), and slightly lower than that of the rTp0326 protein immunization group. However, the reactivity with the lysate of Treponema pallidum was significantly higher than that of the rTp0326 protein immunization group, indicating that intramuscularly delivered rAd5-Tp0326 can induce the production of high-quality specific antibodies.

[0029] 3. Cellular immune detection: Mice were sacrificed on day 42, spleens were isolated, and spleen cell suspensions were prepared. Cells were stimulated with 10 μg / mL rTp0326, and T lymphocyte proliferation was detected by EdU proliferation assay. Cytokine levels were detected by ELISA. The results showed that the T lymphocyte proliferation capacity of the rAd5-Tp0326 intramuscular immunization group was significantly higher than that of the control group (P<0.05), and the IL-17a cytokine level was significantly increased, indicating that the vaccine can induce an effective cellular immune response.

[0030] Example 3

[0031] Efficacy testing of intramuscularly delivered Tp0326 recombinant adenovirus syphilis vaccine 1. Animal grouping and immunization: Adult male New Zealand white rabbits weighing 2.5-3.5 kg were randomly divided into 4 groups of 3 rabbits each: rAd5-Tp0326 intramuscular immunization group, Ad5 (wt) intramuscular immunization group, rTp0326 protein immunization group, and saline control group; rAd5-Tp0326 intramuscular immunization group: 1 mL of 1×10⁻⁶ protein was injected intramuscularly on day 0 and day 21. 8 PFUrAd5-Tp0326 in physiological saline; Ad5 (wt) intramuscular immunization group: 1 mL containing 1×10⁻⁶ PFUrAd5-Tp0326 was injected intramuscularly on day 0 and day 21. 8 PFUAd5 (wt) saline; rTp0326 protein immunization group: 800 μL of adjuvant emulsion containing 200 μg g rTp0326 protein was subcutaneously injected on the back of the neck on day 0 and day 21; control group: 1 mL of saline was injected intramuscularly on day 0 and day 21.

[0032] 2. Challenge Experiment: Three weeks after the last immunization, 1 × 10² strain of Treponema pallidum was injected intradermally into eight sites on the back of rabbits. Skin lesions were observed daily, and blood samples were collected weekly to test TPPA and RPR titers. Rabbits were sacrificed 21 days after challenge, and lesion tissue was collected to test the Treponema pallidum load.

[0033] 3. Results: The mean lesion diameter in the rAd5-Tp0326 intramuscular immunization group was 4.29±0.62 mm, significantly smaller than that in the control group (12.48±0.39 mm, P<0.05). The ulceration rate was 20.8%, significantly lower than that in the control group (75%, P<0.05). The load of Treponema pallidum in the lesion tissue was significantly lower than that in the control group (P<0.05). Furthermore, the RPR titer in the rAd5-Tp0326 intramuscular immunization group was significantly lower than that in the control group, and some rabbits remained RPR negative. This indicates that the intramuscularly delivered Tp0326 recombinant adenovirus syphilis vaccine has excellent protective efficacy and can effectively inhibit Treponema pallidum infection and lesion development.

[0034] Example 4

[0035] Transmission blocking experiment of intramuscularly delivered Tp0326 recombinant adenovirus syphilis vaccine 1. Immunization and Challenge of Donor Rabbits: New Zealand white rabbits were immunized according to the immunization protocol in Example 3. Three weeks after the last immunization, they were intradermally challenged with *Treponema pallidum* strain Nichols, 1×10⁻⁶ oz. per site. 2 A single organism was attacked, and skin lesion tissue was collected 42 days later.

[0036] 2. Recipient rabbit inoculation: The collected lesion tissue was sterilely homogenized and inoculated intratestinally into healthy recipient New Zealand white rabbits. Two recipient rabbits were inoculated in each group with lesion homogenates from donors in the rAd5-Tp0326 intramuscular immunization group, Ad5 (wt) immunization group, and control group, respectively.

[0037] 3. Results Monitoring: Longitudinal monitoring was conducted on recipient rabbits to detect TPPA titers and observe seroconversion. Results showed that neither of the two recipient rabbits vaccinated with donor lesion homogenate from the rAd5-Tp0326 intramuscular immunization group exhibited seroconversion during the entire monitoring period, indicating that the intramuscularly delivered Tp0326 recombinant adenovirus syphilis vaccine effectively blocked the transmission of Treponema pallidum. In contrast, recipient rabbits vaccinated with donor lesion homogenate from the control group and the Ad5(wt) immunization group both showed seroconversion, demonstrating the vaccine's good transmission blocking effect.

[0038] Replenish Figure 2 Preparation and characterization of TP0326 recombinant defective adenovirus: (a) restriction endonuclease digestion analysis for identification of pAdeasy-IRES-TP0326-GFP vector; (b) process of recombinant adenovirus infection of HEK293T cells; (c) process of recombinant adenovirus infection of Sflep cells; (d) analysis of TP0326 protein expression in different cells after infection with recombinant adenovirus by Western blotting; (e) characterization of wild-type and recombinant adenovirus using transmission electron microscopy.

[0039] Replenish Figure 3Differential activation and functional polarization of T cell subsets in mice after TP0326 vaccination: (a) Representative flow cytometry sorting strategy for CD4+ T cell surface activation markers; (b) Intracellular cytokine staining sorting strategy for assessing CD4+ T helper cell polarization; (c) Sorting strategy for intracellular staining of CD8+ T cells using granzyme B and perforin; (d) Quantitative analysis of CD25+, CD62L+, and CD69+ CD4+ T cells, with intracellular staining showing expression of IL-4+, IFN-γ, L-γ, and 17A+ after immunization. CD8+ T cells expressing granzynne B and perforin; (e) T cell proliferation was assessed by EdU inclusion method after in vitro antigen stimulation. Both vaccine groups showed enhanced T cell proliferation compared to the control group, indicating effective T cell activation; (f) Concentrations of interleukin-2 (IL-2), interleukin-17A (IL-17A), tumor necrosis factor (TNF-α), and interferon-γ (IFN-γ) in the serum of each vaccine immunization group; (g) Concentration of IgG2b in the serum of mice in each vaccine group at the immunization endpoint; ns: no significant difference.

[0040] Replenish Figure 4 The in vitro phagocytic activity of serum antibodies against Nichols reference strain and clinical SS14L strain was investigated by immunofluorescence assay. (a) The phagocytic activity of serum antibodies against Nichols reference strain produced by different vaccination regimens was investigated by immunofluorescence assay. (b) The phagocytic activity of serum antibodies against different clinical strains SS14L was investigated by immunofluorescence assay.

[0041] Replenish Figure 5 The in vitro phagocytic activity of vaccine-immunized rabbit serum antibodies against Nichols reference strain and clinical SS14L strain was investigated; (a) the phagocytic activity of different vaccine regimens against Nichols reference strain was detected by immunofluorescence; (b) the phagocytic activity of different clinical SS14L strains against immunized rabbit serum antibodies was detected by immunofluorescence.

[0042] Replenish Figure 6 The characterization of vaccine-induced humoral and cellular immune responses prior to pathogen challenge; (a) purification of IgG from immune sera from different vaccine immunization populations using protein A resin; (b) measurement of serum cytokine levels prior to T. pallidum infection by ELISA at an infection dose of 1 × 10⁻⁶. 5 One bacterial cell.

[0043] Replenish Figure 7 The subcutaneous area of ​​the rabbit's back was vaccinated and subsequently treated with a high dose of Treponema pallidum (1×10⁶ per site).5 Pathological phenotypic changes that occur after a challenge (individual bacteria).

[0044] Replenish Figure 8 (a) Herpes on the back skin of rabbits of different immune groups and results of dark-field examination.

[0045] Enzyme-linked immunosorbent assay (ELISA) was used to detect *T. pallidum* at a concentration of 1 × 10⁻⁶. 2 Serum cytokine levels measured before a dose of live bacteria is administered.

[0046] Replenish Figure 9 The subcutaneous area of ​​the rabbit's back was vaccinated and subsequently treated with a low dose of Treponema pallidum (1×10⁶ per site). 2 Pathological phenotypic changes following challenge with individual bacteria. (a) Herpes lesions on the dorsal skin of different immunized groups and their dark-field examination results.

[0047] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. An intramuscularly delivered Tp0326 recombinant adenovirus syphilis vaccine, characterized in that, include: Recombinant adenovirus rAd5-Tp0326; The recombinant adenovirus rAd5-Tp0326 was constructed by inserting the full-length gene of Treponema pallidum Tp0326 into the adenovirus expression vector pshuttle-IRES-rGFP-1 and homologously recombinating it with the adenovirus backbone plasmid pAdEasy-1. The recombinant adenovirus rAd5-Tp0326 can express the Tp0326 antigen in host cells. The vaccine is delivered via intramuscular injection to induce a specific immune response against Treponema pallidum and inhibit Treponema pallidum infection and transmission.

2. The intramuscularly delivered Tp0326 recombinant adenovirus syphilis vaccine according to claim 1, characterized in that: The Tp0326 gene of *Treponema pallidum* is the full-length coding sequence of Tp0326 in *Treponema pallidum* strain Nichols, and the expressed Tp0326 antigen is a recombinant protein containing an N-terminal 6×His tag.

3. The intramuscularly delivered Tp0326 recombinant adenovirus syphilis vaccine according to claim 1, characterized in that: The method for constructing the recombinant adenovirus rAd5-Tp0326 includes the following steps:

1. The full-length Tp0326 gene was amplified from the genomic DNA of Treponema pallidum by PCR; 2. The amplified Tp0326 gene was cloned into the pshuttle-IRES-rGFP-1 shuttle plasmid through restriction enzyme sites to construct a recombinant shuttle plasmid; 3. The recombinant shuttle plasmid was linearized with restriction endonuclease PmeI and co-transformed with the pAdEasy-1 backbone plasmid into E. coli BJ5183 competent cells for homologous recombination.

4. Screen for positive recombinant adenovirus plasmids, linearize them with restriction endonuclease PacI, transfect HEK293T cells with Lipofectamine®LTX, and package them to obtain recombinant adenovirus rAd5-Tp0326; 5. The recombinant adenovirus rAd5-Tp0326 was amplified in HEK293T cells and purified by CsCl density gradient centrifugation to obtain high-purity recombinant adenovirus particles.

4. The intramuscularly delivered Tp0326 recombinant adenovirus syphilis vaccine according to claim 1, characterized in that: The purified recombinant adenovirus rAd5-Tp0326 titer in step 5 was 1×10⁻⁶. 10 VP / mL, the virus particles have a complete icosahedral morphology.

5. The intramuscularly delivered Tp0326 recombinant adenovirus syphilis vaccine according to claim 1, characterized in that: The intramuscular delivery dose of the vaccine is as follows: for New Zealand white rabbits, a single intramuscular injection of 1 mL, containing 1×10⁻⁶ mmol / L. 8 PFU recombinant adenovirus rAd5-Tp0326; for BALB / c mice, a single intramuscular injection of 50 μL containing 1×10 7 PFU recombinant adenovirus rAd5-Tp0326.

6. The intramuscularly delivered Tp0326 recombinant adenovirus syphilis vaccine according to claim 1, characterized in that: The immunization schedule for the vaccine consists of two intramuscular injections, with a 21-day interval between the initial and booster immunizations, and the same dosage for both immunizations.

7. The intramuscularly delivered Tp0326 recombinant adenovirus syphilis vaccine according to claim 1, characterized in that: The specific immune response induced by the vaccine includes: producing high-titer anti-Tp0326 IgG antibodies, inducing a Th2-biased immune response (IgG1 / IgG2a>1), promoting T lymphocyte antigen-specific proliferation, and increasing IL-17a cytokine levels.

8. The intramuscularly delivered Tp0326 recombinant adenovirus syphilis vaccine according to claim 1, characterized in that: The specific antibodies induced by the vaccine can inhibit the adhesion of Treponema pallidum to cottontail rabbit epithelial Sf1Ep cells and promote the opsonization of Treponema pallidum strains Nichols and SS14 by mouse bone marrow-derived macrophages or rabbit peritoneal macrophages.

9. The intramuscularly delivered Tp0326 recombinant adenovirus syphilis vaccine according to claim 1, characterized in that: The vaccine is used to prevent infection with Treponema pallidum, and can reduce the development of skin lesions after infection, reduce the viral load of Treponema pallidum in the lesion tissue, and block the transmission of Treponema pallidum between hosts.

10. The intramuscularly delivered Tp0326 recombinant adenovirus syphilis vaccine according to claim 1, characterized in that: After the recombinant adenovirus rAd5-Tp0326 infected HEK293T cells and Sf1Ep cells, it was able to effectively express the Tp0326 antigen. The expressed antigen was verified by Western blot, and a specific band appeared at the expected molecular weight.

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