Advancements in the development of nucleic acid vaccines for syphilis prevention and control

ABSTRACT

Syphilis, a chronic systemic sexually transmitted disease, is caused by the bacterium Treponema pallidum (T. pallidum). Currently, syphilis remains a widespread infectious disease with significant disease burden in many countries. Despite the absence of identified penicillin-resistant strains, challenges in syphilis treatment persist due to penicillin allergies, supply issues, and the emergence of macrolide-resistant strains. Vaccines represent the most cost-effective strategy to prevent and control the syphilis epidemic. In light of the ongoing global coronavirus disease 2019 (COVID-19) pandemic, nucleic acid vaccines have gained prominence in the field of vaccine research and development, owing to their superior efficiency compared to traditional vaccines. This review summarizes the current state of the syphilis epidemic and the preliminary findings in T. pallidum nucleic acid vaccine research, discusses the challenges associated with the development of T. pallidum nucleic acid vaccines, and proposes strategies and measures for future T. pallidum vaccine development.

Introduction

Syphilis, caused by Treponema Pallidum (T. pallidum), is a multistage sexually transmitted infectious diseases which poses a serious threat to global public health.¹ The course of T. pallidum infection is divided into early and late stages, with two years as the boundary. According to the clinical manifestations and incubation period of different course of the disease, it is further divided into primary, secondary, and tertiary syphilis.^2–4 Primary syphilis typically presents as a single painless ulcer or chancre at the site of infection but can also manifest as multiple, atypical, or painful lesions. Secondary syphilis can involve skin rash, mucocutaneous lesions, and lymphadenopathy. Tertiary syphilis may present with cardiac involvement, gummatous lesions, tabes dorsalis, and general paresis.⁵

The World Health Organization estimates that between 5.6 million and 11 million new cases of syphilis occur globally each year, while the global prevalence of the disease ranges between 18 million and 36 million.⁶ The estimated prevalence and infection rate of syphilis vary across regions or countries. Africa has the highest prevalence, with over 60% of new cases occurring in low-income and middle-income countries (LMICs).⁷ Europe, the United Kingdom, the United States, Canada and China also have a greater risk of syphilis infection.⁸ In recent years, the incidence of congenital syphilis infection has been rising in countries of different economic levels. Notably, congenital syphilis cases increased to 11.6 per 100,000 live births in the United States in 2014, the highest level since 2001, while the increase was even more pronounced in China, where 69.9 per 100,000 live births were reported in 2013.⁹

The study led by the London School of Hygiene and Tropical Medicine, London, UK, and published in The Lancet Global Health, presents the first global syphilis prevalence estimate among men who have sex with men (MSM). Findings from this global review reveal that MSM have a high burden of T. pallidum infection, with significant variation across countries.¹⁰ Syphilis rates among MSM have been rising sharply in the United States, increasing from 15.8 per 100,000 MSM in 2000 to 228.8 per 100,000 MSM in 2013.¹¹ In addition, the pathogenesis of T. pallidum is varied, such as irregular sexual intercourse, blood contact, and sharing intimate household items. It is well established that T. pallidum contributes to the acquisition and transmission of HIV-1 by providing a breach in the host integument, and more specifically by increasing the expression of the viral coreceptor CCR5 on macrophages present in T. pallidum lesions.¹² Symptomatic T. pallidum infection is estimated to increase HIV transmission and acquisition by 2 to 5 times.¹³ The global public health threat posed by syphilis underscores the need for effective syphilis control measures.

Treatment strategies for syphilis

Currently, penicillin is the most effective antimicrobial agent for treating syphilis.¹⁴ Although T. pallidum has not yet shown resistance to penicillin, its interaction with autoimmune mechanisms could potentially create an environment more favorable for T. pallidum growth, highlighting the need for alternative prevention and control strategies.^1,15 Simultaneously, due to the devastating consequences of congenital syphilis, penicillin-allergic pregnant women require desensitization before penicillin treatment, which further emphasizes the need for alternative control measures.^16–18 Vaccination is the most cost-effective public health intervention to prevent and control infections. A safe and effective syphilis vaccine could drastically reduce the global burden of syphilis disease and potentially lead to syphilis elimination worldwide.¹⁹

An overview of the current vaccine techniques

Traditional vaccine technology

As is well known, existing traditional vaccines developed include inactivated, attenuated, and subunit vaccines, each with their advantages and disadvantages.²⁰ Inactivated vaccines exhibit high safety and provide comprehensive protection, but they require multiple injections.²¹ Attenuated vaccines demonstrate strong immunogenicity and induce robust humoral and cellular immunity, but they carry the risk of reverting to high virulence after administration.²² Subunit vaccines offer high safety but exhibit poor immunogenicity, which can induce humoral immunity and weak cellular immunity, providing only partial protective effects.²³ Importantly, with the continuous development of the vaccine field, nucleic acid vaccines have emerged as a more flexible alternative compared to traditional vaccines.²⁴ At present, nucleic acid vaccines have been successfully applied in the prevention of various animal diseases. Although there is only one approved nucleic acid vaccine for human infectious diseases, numerous nucleic acid vaccines, including those for cancer, are currently under development and show promise for future applications. With the improvement of medical level, people pay more attention to the prevention of diseases, and there are many nucleic acid vaccines under development.²⁵ Therefore, developing a syphilis nucleic acid vaccine to prevent and control syphilis infection is a highly feasible approach.

Nucleic acid vaccine technology

DNA vaccine

In 1990, Wolff et al. injected a DNA recombinant expression vector into the skeletal muscle of mice and unexpectedly discovered that the genes on the vector were expressed in local muscle cells, leading to the production of antibodies. This groundbreaking finding marked the birth of DNA vaccines (nucleic acid vaccines).²⁶ In simple terms, a DNA vaccine is a novel genetically engineered vaccine that introduces a plasmid encoding the target antigen protein gene sequence into host cells through intramuscular injection, allowing the antigen protein to be expressed by the transcription system and inducing the host immune response against the antigen protein.²⁷ Over the years, researchers have made significant progress in addressing challenges related to the development of DNA vaccines for the prevention and treatment of certain infectious diseases, as well as cancer. However, it is important to acknowledge that many subunit vaccines have already shown high levels of protection against various infectious diseases, such as influenza and hepatitis, and even cancer.²⁸ DNA vaccines offer several advantages over traditional vaccines: DNA vaccines can encode various types of genes, including viral or bacterial antigens, as well as immune and biological proteins, showcasing high flexibility. Additionally, DNA vaccines are stable, easy to store and transport, and can be mass-produced.^29,30 This wealth of theoretical and practical knowledge provides a solid foundation for the research and development of syphilis DNA vaccines.

RNA vaccine

DNA vaccines offer significant benefits in terms of rapid production, but they also pose potential risks, such as insertional mutations, genomic integration, and immune tolerance.³¹ Interestingly, mRNA vaccines have been shown to effectively circumvent some of these safety concerns.³² In particular, the ongoing global COVID-19 pandemic has highlighted the critical role of messenger RNA (mRNA) vaccines in the prevention and control of infectious diseases.^33,34 As a result, mRNA vaccines have become a focal point in the field of vaccine research and development, owing to their increased efficiency compared to traditional vaccines and the interest in nucleic acid vaccines.³⁵ mRNA vaccines can be classified into two types: conventional mRNA vaccines and self-amplifying mRNA vaccines. Both types can translate and process targeted antigens in the cytoplasm, inducing adaptive immune responses through host cell translation mechanisms.³⁶ Importantly, mRNA vaccines do not enter the nucleus, produce infectious particles, or integrate into the host cell genome. Additionally, the transient expression of mRNA encoding the target antigens allows for better control of antigen exposure, reducing the risk of tolerance induction associated with prolonged antigen exposure.³⁷ Given these core principles and features, mRNA vaccines are hypothesized making it an effective strategy to prevent the spread to be highly safe. Since the pandemic of COVID-19 in 2019, mRNA vaccines have become the most promising vaccine candidates to fight the epidemic due to their rapid production time, cost-effectiveness, versatility in vaccine design, and clinically proven ability to induce cellular and humoral immune responses.^38,39 Furthermore, the cell- and egg-free manufacturing process makes mRNA vaccines promising candidates, with the potential to bridge the gap between emerging epidemics, infectious diseases, and the urgent need for effective vaccines.³⁶

Study on syphilis nucleic acid vaccine

Syphilis DNA nucleic acid vaccine results

As DNA vaccine research and development technology continues to mature, early studies on T. pallidum DNA vaccines have also delved deeper into DNA vaccine investigations. Zhao et al. prepared pcDNA/Tp92, pcDNA/Gpd, pcDNA/IL-2, and pcDNA/Gpd-IL-2 gene vaccines, along with additional CS nanoparticle-coated nucleic acid vaccines.⁴⁰ Researchers evaluated vaccine titers using multipoint muscle injection, the MTT method, and ELISA. The study revealed that the combined immune vaccine group exhibited greater immune activity and protection than the single gene vaccine group, but the CS nanoparticle packaging did not optimize the vaccine. Additionally, the investigators discovered that after initial immunization with pcD/Gpd-IL-2 vaccine muscle injection, nasal administration of CpGODN + Gpd-IL-2 protein resulted in an immune response not significantly different from that of the pcD/Gpd-IL-2 muscle injection immunization group, and could also stimulate a higher mucosal immune effect.⁴¹ Zheng Kang et al. intramuscularly injected plasmid DNA encoding flagellin pcDNA3/flab3 into New Zealand rabbits and observed a significant specific antibody response in the immunized rabbits. INF-γ and CD8+ were significantly up-regulated, and high levels of IL-6 and IL-8 were detected.⁴² Concurrently, the study demonstrated that the immunogenicity of flagellin plasmid DNA was significantly superior to that of the recombinant flagellin vaccine, suggesting it might be an effective vaccine strategy to prevent the spread of T. pallidum. Existing studies have found that although DNA vaccine immunization can induce the production of high levels of antibodies, the cellular immune response is relatively weak, resulting in a suboptimal immune effect. To date, no T. pallidum DNA vaccine with robust protective efficacy has been identified.

T. pallidum is generally considered an extracellular pathogen with the ability to evade recognition and circumvent certain host immune responses.¹ In order to design and develop a safe and effective mRNA vaccine against T. pallidum, it is worth considering research on mRNA vaccines targeting Group A Streptococcus (GAS, Streptococcus pyogenes) and Group B Streptococcus (GBS, Streptococcus agalactiae), which also belong to extracellular pathogens. Maruggi G et al assessed the efficacy of self-amplifying mRNA (SAM) vaccines expressing antigenic proteins based on GAS and GBS in mice.⁴³ They found that both vaccines not only induced antibody responses providing consistent immune protection against GAS and GBS infections in mice but also elicited primarily TH1-like immune responses. Notably, TH1-like immune responses are advantageous for eliminating T. pallidum and preventing bacterial infections. However, compared to recombinant proteins, particularly for BP-2a, the overall protection induced by SAM vaccines was inferior in magnitude.⁴⁴ Interestingly, an mRNA vaccine targeting Mycobacterium tuberculosis employed TLR4 agonist (RpfE) peptide as an adjuvant to enhance the immunogenicity of the vaccine.^44,45 TLR4 is a well-known receptor that plays an essential role in recognizing M. tuberculosis, activating macrophages and dendritic cells to induce innate and adaptive immunity.^45,46 Additionally, enhancing the immunogenicity and optimizing the efficiency of an mRNA vaccine can be achieved by selecting dominant antigens, modifying the mRNA sequence, using appropriate adjuvants, employing a suitable delivery system, and determining the optimal immunization route.⁴⁷ Immunoinformatics, which has been widely used to develop vaccines against pathogens including Salmonella, Rickettsia, and Staphylococcus aureus, may be an effective approach to engineer mRNA vaccines.^46,48,49 Currently, there is no licensed mRNA vaccine against T. pallidum, but the undeniable fact is that the wealth of practical experience in the development of mRNA vaccines against viruses and other infectious pathogens can be beneficial.

Challenges in T. pallidum nucleic acid vaccine research

In practical studies, T. pallidum DNA vaccine research faces numerous challenges. ① Currently, T. pallidum is primarily obtained from rabbit testes, but the procedure is complex, the vaccination success rate is inconsistent, and purification is difficult, hindering syphilis research. Edmondson et al. successfully cultured and propagated T. pallidum strains in vitro while preserving the wild-type morphology, activity, and virulence of the strain.^50,51 Although the in vitro culture conditions for T. pallidum are demanding and obtaining large quantities of spirochetes is challenging, it provides a foundation for the development of T. pallidum DNA vaccines.^52,53 ② T. pallidum gene editing is in its infancy, making it difficult to investigate the function of each gene product.⁵⁴ However, Borrelia burgdorferi shares similarities with T. pallidum in genome and outer membrane structure, and transferring T. pallidum genes into B. burgdorferi can express the gene product and its function. To date, TprK, Tp0435, and Tp0136 proteins have been successfully expressed by nonpathogenic Borrelia burgdorferi strain B314.⁵⁵ This expression system eliminates interference from other proteins in gene expression products,⁵⁶ and can be employed not only for the study of T. pallidum gene function but also for the prediction and evaluation of vaccine targets. ③ Selecting objective genes is difficult: T. pallidum has few outer membrane proteins and high variability among strains, such as tprK and tp0136.^57,58 Identifying amino acid sequences that are highly conserved among different strains and whose coding products can induce strong cellular and humoral immune responses proves to be challenging. However, through the cloning and expression of recombinant T. pallidum DNA and the investigation of the structure and function of various T. pallidum protein antigens, the physicochemical properties and gene structure of multiple T. pallidum primary lipoprotein antigens are becoming increasingly clear, greatly facilitating the study of candidate gene screening for T. pallidum DNA vaccines. Additionally, there are several unresolved issues, such as safety, immune tolerance, and the challenge of regulating antigen expression after entering the body.

Recommendations for T. pallidum nucleic acid vaccine development

Current research indicates that although DNA vaccines can induce high levels of antibodies, they often elicit weak cellular immunity, resulting in suboptimal immune efficacy.⁴⁰ To date, no T. pallidum DNA vaccine with robust protective effects has been identified. This suggests that future research should concentrate on advancing key nucleic acid vaccine technologies, such as enhancing vaccine carriers, optimizing delivery systems, augmenting immune responses, improving mRNA stability and survivability, and selecting appropriate T. pallidum antigens.

Selection of the vaccine vectors

Plasmid Vector: Contemporary T. pallidum DNA vaccine design typically employs nucleic acid synthesis and potential one-step cloning into plasmid vectors, thereby reducing manufacturing costs and time.⁵⁹ Plasmid DNA is highly stable at room temperature, decreasing the reliance on cold chain logistics during transportation.⁶⁰ Utilizing DNA plasmids for vaccination circumvents the need to purify proteins from infectious pathogens, enhancing safety.^59,61

Chitosan Nanoparticles (CS) Carrier: Chitosan nanoparticles can deliver biologically active materials into cells without compromising the integrity of the cargo or the cell. These nanoparticles are internalized into cells via endocytosis, ensuring efficient delivery.^60,62

Bacterial Ghost (BG): BGs are cytoplasm-free, non-denatured Gram-negative bacterial cell envelopes. They offer significant advantages over other engineered biological delivery particles due to their inherent cell and tissue affinity, ease of production, and storage capabilities that do not require refrigeration.^60,62 BGs possess a strong capacity for loading and delivering foreign DNA and recombinant protein antigens, inducing high-level immune responses to the loaded antigens.

Liposomes: spherical vesicles composed of phospholipids and cholesterol can not only improve the transfection efficiency, but also have the adjuvant effect.^62,63

Improve the vaccine delivery system

Like more traditional protein-based vaccines, DNA can be delivered through various methods, including intramuscular (IM), intradermal (ID), mucosal, or transdermal delivery.^59,61 Different immune pathways involve distinct APCs and antigen presentation methods, resulting in diverse types and intensities of immune responses.⁵⁹ With the continuous development of DNA vaccines, numerous vaccine delivery methods have emerged:

Mechanical delivery methods such as intramuscular or intradermal electroporation: Electroporated patients in a study of the HPV DNA vaccine demonstrated increased multifunctional antigen-specific CD8+ T cells.⁶¹

Using physical forces such as particle bombardment (gene gun): Delivery of DNA plasmids into target tissues or cells can produce sustained antibody titers in subjects who have previously failed to respond to approved subunit vaccines.⁶⁴

DNA vaccine delivery can also be accomplished using biodegradable polymer particles and nanoparticles composed of amphiphilic molecules, ranging in size from 0.5–10 µm.⁵⁹ Microparticles and nanoparticles can protect plasmid DNA from nuclease degradation and promote the continuous release of the vaccine. Particles can be delivered orally or via intraperitoneal routes, allowing direct transfection of dendritic cells (DCs), thereby increasing DC activation.⁶⁵ Currently, some studies have identified a new immunization strategy, multi-channel combined immunization, which can achieve better immune effects through mutual reinforcement. The method of primary immunization with T. pallidum DNA vaccine combined with protein immunization can effectively induce systemic and mucosal humoral immune responses in mice. Primary immunization followed by multiple intramuscular injections can produce a stronger immune response and prolong the maintenance of protective immunity.⁴⁰

Utilization of immunopotentiators and adjuvants

The employment of immunopotentiators and adjuvants, such as cytokine genes, including Interleukin (IL)-2, IL-12, and IL-15, has been demonstrated to markedly augment both humoral and cell-mediated immune responses upon co-administration with DNA vaccines.^66,67 Furthermore, Toll-like receptor (TLR) ligands, specifically targeting TLR-3 and TLR-9, have been proven to enhance immunological outcomes.⁶⁸ For instance, the incorporation of a CpG adjuvant has been shown to bolster the mucosal immunogenicity and overall efficacy of a T. pallidum DNA vaccine in a rabbit model.⁴¹

Enhancement of mRNA stability and accessibility

In-depth investigations have been conducted on preserving the activity and shelf life of mRNA vaccines. The design of synthetic mRNA elements, such as the 5’ untranslated region (UTR), 3’ UTR region sequence, poly-A tail, cap, and nucleoside triphosphate (NTP), is optimized during in vitro transcription on the DNA template.^69,70 Alternatively, separation and purification technologies can be employed to improve mRNA stability and translation efficiency.⁶⁹ Further advancements in delivery systems and the utilization of effective mRNA delivery vectors can substantially enhance the stability and translation efficacy of mRNA vaccines. Research on the storage effects of mRNA complexed with carrier molecules under freezing conditions aims to prolong the preservation period of vaccines.⁷¹

Selection of the vaccine antigens

Dr. James Miller et al. showed that freshly-isolated, -/-irradiated treponemes immunized animals produced immunity stronger than those obtained by treponemal immunity after 6 to 10 days of refrigeration. This may be due to freshly isolated organisms indicating the presence of an important immunogen.⁷² The identification of suitable antigens is a critical and fundamental step in vaccine development.^71,73 The clearance of T. pallidum primarily occurs through the induction of inflammatory cytokines by multiple T. pallidum antigenic clusters, including flagellin and lipoproteins, which activate macrophages and stimulate T and B lymphocytes, ultimately leading to opsonophagocytosis.^73–75 The cloning and genetic expression of recombinant T. pallidum DNA and the ongoing elucidation of the structure and function of various T. pallidum protein antigens have significantly advanced our understanding of their properties and gene structure, thus facilitating the screening process for T. pallidum DNA vaccines. In numerous other studies, the protective capacity of T. pallidum molecules (e.g., Tp0751, Gpd, TmpB, Tp92, TpN15, TpN47, TprF, TprI, TprK) against infectious challenges has been assessed.^9,76 We summarize the recent studies of syphilis protein by various researchers to provide a reference for the selection of candidate antigens (Table 1).

Table 1.

Summary of T. pallidum protein research.

Number	Name	Functional or predicted function	Protective capacity
TP genes
TP0009	tprA	PUFa	PUF
TP0011	tprB	PUF	PUF
TP0117	OMP (tprC)	PUF	Maybe
TP0131	OMP (tprD)	PUF	Maybe
TP0313	tprE	PUF	PUF
TP0316	OMP (tprF)	Initiates a significant antibody response77,78	Sure
TP0317	tprG	PUF	PUF
TP0610	tprH	PUF	PUF
TP0620	OMP、tprI (Porin)	Initiates a significant antibody response77,78	Maybe
TP0621	tprJ (Potential porin)	PUF	PUF
TP0897	tprK (rare OMP)	Related with antibody variants and immune escape79–81	Sure
TP1031	tprL (Potential porin)	conserved between syphilis strains82	Maybe
Hemolysins83,84
TP0027	hlyA	PUF	PUF
TP0028	hlyB
TP0694	tlyC
TP0936	hlyC
TP1037	hlyIII
OMPb
TP0034	rare OMP (TroA)	substrate-binding proteins (SBPs) for ABC transporters85	Maybe
TP0155	fibronectin-binding proteins	preferentially bound the matrix form of fibronectin86–88	Maybe
TP0126	putative OMP	structural homology with OmpW;limited immunogenicity89	Maybe
TP0134	HP,putative OMP	PUF	PUF
TP0136	Fibronectin-binding protein	Adhesive to colonization,production of protective antibodies90,91	Maybe
TP0257	OMP (Gpd)	highly conserved among different strains, producing strong partial protective immunity92,93	Sure
TP0326	OMP (Tp92)	inflammatory response, inducing death of numerous immune response cells;highly conservative among different strains,producing strong partial protective immunity(opsonic and enhanced the phagocytocis of Tp by macrophages)92–94	Sure
TP0435	lipoprotein、adhesin (Tp17)	Adhesive colonization, producing surface and periplasmic immunogenic lipoprotein isoforms; producing a strong humoral response1,95–97	Sure
TP0453	OMP	By interfering with the membrane and promoting TM solute flux, it is speculated that TP0453 may make the globus pallidus OM permeable to multiple nutrients93	Sure
TP0462	putative OMP	PUF	PUF
TP0483	fibronectin-binding proteins	bound both the soluble and matrix forms of fibronectin98,99	Maybe
TP0548	Predicted rare OMP	PUF	PUF
TP0574	OMP (TpN47)	Binding to the pattern receptor, activating endothelial cells and innate immune cells, adhering to the host, causing a strong specific immune response100	Sure
TP0663	rare OMP	high specificity and sensitivity for the early and late Tp examinatio91	Maybe
TP0684	OMP	Binding to the pattern receptor, activating endothelial cells and innate immune cells, adhering to the host, causing a strong specific immune response101	Sure
TP0693	putative OMP	Secreted proteins with strong immunogenicity have high specificity and sensitivity to all stages of Tp examination102	Sure
TP0733	HP,putative OMP	PUF	PUF
TP0750	adhesin	Similar structure to TP0751 structure103	Maybe
TP0751	adhesin	Adhesive to colonization and production of protective antibodies17	Sure
TP0768	OMP (TmpA)	As a serological examination method of envelope antigen, high sensitivity and high specificity104,105	Sure
TP0821	lipoprotein	High immunogenic and high immunoreactivity, triggers delayed hypersensitivity, induces specific cellular immune response1,106,107	Sure
TP0858	Predicted OMP	PUF	PUF
TP0865	HP,putative OMP	PUF	PUF
TP0965	synexin (Membrane fusion protein)	Strong immunogenic and immunoreactivity, activating endothelial cells, leading to endothelial barrier dysfunction, may function in late stages108,109	Sure
TP1038	OMP (TpF1)	produces an inflammatory response that induces a regulatory T (Treg) response and stimulates the monocyte release of IL-10 and TGF-β110	Sure
TP1046	OMP	PUF	PUF
flagellin protein
TP0868	FlaB1	PUF	PUF
TP0792	FlaB2	PUF	PUF
TP0870	FlaB3	Have a strong immunogenicity, effectively stimulate the body’s humoral, cell immunity111	Sure
FlaA		PUF	PUF
methylaccepting chemotaxis proteins
TP0040	MCP1	bind exogenously derived ligands within the periplasm and relay chemotactic signals to the more distal flagellar motors; cause humoral reactions112,113	Sure
TP0488	MCP2
TP0639	MCP3
TP0640	MCP4
ABC transporters and symporters
TP0119-TP0120-TP0821	TpN32	uptake of methionine114	PUF
TP0585	oligopeptides (OppA)	corresponding permeases and ATP-binding proteins have yet to be identified
TP0308	histidine (His)
TP0309	polar amino acids
TP0144	TbpAPQ (TP0142–0144)	the SBP for a thiamine ABC transporter115
other
TP0225	putative leucine-rich repeat (LRR)	PUF	PUF

^aPUF: unknown.

^bOMP: outer member protein.

Discussion

The understanding of immune correlates in humans remains limited. It is widely acknowledged that individuals who infect T. pallidum can be re-infected following treatment, and this cycle may recur multiple times. Human studies have demonstrated that individuals with late latent syphilis exhibit resistance to symptomatic reinfection with a heterologous strain of T. pallidum, whereas those in earlier stages display evidence of infection upon challenge. This observation aligns with the prolonged immunization period required to induce protection in Miller’s successful vaccine. The development of immunity observed in rabbits exhibits components of subspecies- and even strain-specificity, which are most likely attributable to antigenic differences among strains. Consequently, T. pallidum vaccine development efforts should encompass evaluations of extended immunization schedules. Moreover, the selection of immunogens must account for antigenic diversity among strains and address the effects of antigenic variation on immune evasion.

Vaccination against syphilis not only reduces global morbidity and mortality, healthcare costs and overall burden of disease for syphilis, but also the importance of T. pallidum vaccines in eliminating congenital syphilis, which can significantly reduce infant mortality, prevent long-term complications and improve maternal health. The production and application of T. pallidum vaccines can emphasize the prevention of the spread of disease in family planning, preconception and prenatal care, and the impact of the disease on future generations and on the overall health of families. It is well known that infection with T. pallidum reduces the body’s autoimmunity and is complicated by the occurrence of many autoimmune diseases. The importance of T. pallidum vaccines in addressing cross-infection or co-infection problems such as syphilis and HIV by reducing the risk associated with co-infection and improving the overall management of sexually transmitted infections.

Funding Statement

This work was supported by the Hunan clinical medical technology innovation guidance project [Grant numbers 2021SK50402], [82101869], National Natural Science Foundation of China [Grant numbers 81702046 and 82002182], Natural Science Foundation of Hunan Province [Grant numbers 2021JJ40479 and 2019JJ50535], Hunan Province Cooperative Innovation Center for Molecular Target New Drug Study [grant number 2015–351], HunanProvincial Key Laboratory for Special Pathogens Prevention and Control Foundation [grant number 2014–5].

Disclosure statement

No potential conflict of interest was reported by the author(s).