Lorenzo Giacani

Syphilis continues to be a significant global health concern. To define the extent of the IgG and IgA antibody reactome in patients with syphilis and identify reactive antigens and reactivity patterns that could facilitate early diagnosis and possibly serve as biomarkers for treatment response and staging, we developed a pan-proteomic microarray carrying 98.8% (1,009 antigens) of the annotated polypeptides of two Treponema pallidum subsp . pallidum strains (Nichols and SS14). In this study, we probed the array with 217 sera collected pre- and post-treatment from 122 syphilis patients to detect IgG and IgA antibodies. Although a minimal IgA response to T. pallidum antigens were detected in these sera, with only 21 reactive targets on the array, corresponding to 20 T. pallidum antigens, significant IgG reactivity was detected to a total of 104 array protein spots, corresponding to 101 T. pallidum antigens, and roughly encompassing 10% of the pathogen’s proteome. No targets were differentially recognized in pre-treatment sera when factoring in covariates such as syphilis stage, syphilis history, and human immunodeficiency virus status, but a significant decrease in reactivity to 82 antigens was detected in the sera collected post-treatment compared to sera collected at diagnosis. This work helped define the extent of the IgG and IgA antibody response in syphilis patients and identified antigens that could be further evaluated to improve treponemal serological tests. Antigens inducing a detectable IgA response in adults, albeit very few, could be further investigated as diagnostic markers. IMPORTANCE: This investigation improved our understanding of the IgG and IgA humoral response to virtually all proteins of Treponema pallidum subsp. pallidum ( T. pallidum ) during natural infection in patients with active and treated syphilis. Using samples collected pre- and post-treatment from individuals presenting with syphilis at different stages, human immunodeficiency virus status, and history of recurrent infection, we identified antigens that could be further investigated to improve early syphilis and congenital syphilis diagnosis and serve as possible biomarkers to monitor treatment response.

Treponema pallidum subsp. pallidum , the causative agent of syphilis, remains difficult to study owing to long-standing limitations in in vitro cultivation. Although a rabbit epithelial cell co-culture system (Sf1Ep) enabled major advances, the absence of human cell-based models limits clinical relevance and mechanistic understanding of host–pathogen interactions. Here, we established a co-culture system using human epithelial cell lines capable of supporting T. pallidum growth in vitro. Six epithelial or epithelial-like cell lines from diverse tissues were evaluated under microaerophilic conditions using standard cultivation medium. CAL-39 (vulva) and HepG2 (liver) supported T. pallidum survival, replication, characteristic growth behaviours, and long-term passage at levels comparable to Sf1Ep cells. Growth kinetics, attachment dynamics, and motility were quantified over extended culture. Using live-cell imaging, we defined two distinct host-cell interaction behaviours: surface-associated crawling and stable single-polar attachment. Both behaviours were observed across all tested cell lines but persisted only in those permissive for sustained growth. Together, our findings establish clinically relevant human epithelial co-culture models for T. pallidum , provide new insights into host-cell-dependent growth and motility, and create a platform for mechanistic studies of syphilis pathogenesis and vaccine target discovery.

In the last decade, we have witnessed outstanding scientific advances towards the development of a globally efficacious syphilis vaccine. This manuscript summarizes information presented at the Shanghai International Syphilis Symposium (October 20–22, 2025, Shanghai, China) during the Vaccine Development session. Dr. Lorenzo Giacani (University of Washington) moderated the session. Dr. Justin Radolf (UConn Health) summarized key historical research findings that inform modern vaccine development, emphasizing how protective immunity was demonstrated in the early days of syphilis vaccine research and attributed to antibodies. Drs. Caroline Cameron (University of Victoria), Melissa Caimano (UConn Health), and Feijun Zhao (University of South China) discussed their research relating to antigen selection, identification of potentially protective B cell epitopes, results of immunization/challenge experiments, vaccine delivery platforms, and manufacturing requirements, with particular emphasis on the development of chimeric vaccine candidates. While challenges remain, the application of high-throughput immunological, proteomic, and genomic techniques discussed here has revealed surface-exposed epitopes of TPA outer membrane proteins with promising, but as yet unproven, vaccine potential. Multiple vaccine designs have emerged as options to efficiently and effectively package these epitopes in chimeric structures and platforms that can meet manufacturing standards and requirements. Efforts are also ongoing to identify and procure clinically approved adjuvants to be delivered alongside vaccine candidates.

We demonstrated no differences in susceptibility to penicillin G and ceftriaxone in 3 modern Treponema pallidum isolates (UW244B, UW249B, and UW330B), each carrying a variant of the penicillin-binding protein Tp0705. This suggests that these polymorphisms should not be a reason for concern when β-lactams are prescribed for syphilis treatment.

The global resurgence of treponematoses, particularly syphilis, poses a growing public health challenge. Despite recent advances in sequencing technologies, obtaining complete Treponema pallidum genome sequences for epidemiological studies remains time-consuming and challenging due to the difficulty related to procuring clinical samples with sufficient treponemal burden to fulfil the sequencing requirements. There is an urgent need for rapid, cost-effective and accessible typing methods suitable for laboratories with Sanger sequencing resources. Based on the analysis of 121 T. pallidum genomes from geographically diverse regions, we selected seven highly variable genes to form the basis of this new typing system. These seven genes show high discrimination capacity, identifying many allelic profiles among T. pallidum isolates. Importantly, the scheme employs a single-step PCR protocol for the amplification and sequencing of all seven targets enabling straightforward implementation in standard laboratory settings. The MLST was validated using a diverse set of T. pallidum clinical samples from across the globe. A significant proportion of the tested samples showed macrolide resistance, emphasizing the need for epidemiological surveillance. Utilizing this new tool, we have analyzed the genetic variation within and between populations of T. pallidum , considering the geographical origin of the samples. Population structure analysis revealed distinct genetic clusters, underlining complex transmission dynamics of T. pallidum , shaped by local epidemiological factors. The MLST scheme is publicly accessible through the PubMLST database, encouraging widespread adoption in standard laboratories due to this database being user-friendly, intuitive, and fast to implement. The novel MLST scheme offers a promising tool to advance the study of the molecular epidemiology of T. pallidum , facilitate tracking transmission, and establish a global surveillance network with the overall goal of strengthening public health interventions for syphilis control.

Background/Objectives: Syphilis, a chronic sexually transmitted disease caused by the spirochete Treponema pallidum subspecies pallidum (T. pallidum), is still endemic in low- and middle-income countries and has been resurgent for decades in many high-income nations despite being treatable. Improving our understanding of syphilis pathogenesis, immunology, and T. pallidum biology could result in novel measures to curtail syphilis spread, including new therapeutics, a preventive vaccine, and, most importantly, improved diagnostics. Methods: Using overlapping synthetic peptides spanning the length of the T. pallidum Tp0435 mature lipoprotein, an abundant antigen known to induce an immunodominant humoral response during both natural and experimental infection, we evaluated which Tp0435 linear epitopes are most significantly recognized by antibodies from an infected host. Specifically, we used sera from 63 patients with syphilis at different stages, sera from non-syphilis patients (n = 40), and sera longitudinally collected from 10 rabbits infected with either the Nichols or SS14 isolates of T. pallidum, which represent the model strains for the two known circulating clades of this pathogen, to further evaluate the use of this animal model for syphilis studies. Recognized amino acid sequences were then mapped to the experimentally determined Tp0435 structure. Results: Reactive epitopes in both serum groups mapped predominantly to the α-helix preceding Tp0435 soluble β-barrel and the loops of the barrel. Conclusions: In the current effort to improve current syphilis diagnostics, the peptides corresponding to these immunodominant epitopes could help develop epitope-based assays such as peptide-based ELISAs and lateral flow point-of-care tests to improve the performance of treponemal tests and expedite diagnosis in low-income settings, where the infection is still a significant concern for public health and access to facilities with laboratories equipped to perform complex procedures might be challenging.

The recently discovered methodologies to cultivate and genetically manipulate Treponema pallidum subsp. pallidum (T. pallidum) have significantly helped syphilis research, allowing the in vitro evaluation of antibiotic efficacy, performance of controlled studies to assess differential treponemal gene expression, and generation of loss‐of‐function mutants to evaluate the contribution of specific genetic loci to T. pallidum virulence. Building on this progress, we engineered the T. pallidum SS14 strain to express a red‐shifted green fluorescent protein (GFP) and Sf1Ep cells to express mCherry and blue fluorescent protein (BFP) for enhanced visualization. These new resources improve microscopy‐ and cell sorting–based applications for T. pallidum, better capturing the physical interaction between the host and pathogen, among other possibilities. Continued efforts to develop and share new tools and resources are required to help our overall knowledge of T. pallidum biology and syphilis pathogenesis reach that of other bacterial pathogens, including spirochetes.