Abstract Background Syphilis cases continue to increase rapidly in the US. Molecular detection of the pathogen, Treponema pallidum (TP) may speed turnaround time (TAT) and resolve ambiguous results from current diagnostics methods, which require stepwise serologic testing. Here we describe validation and clinical performance of a loop-mediated isothermal amplification (LAMP) assay for TP. Methods Candidate LAMP primers targeting the 23S rRNA locus of TP were evaluated and the best performing set was used for clinical validation per Clinical Laboratory Standards Institute guidelines. We determined the sensitivity, specificity, precision, accuracy, and reproducibility of the TP-LAMP assay in fresh and formalin-fixed tissue, body fluids, and whole blood using contrived specimens spiked with intact treponemes and residual clinical specimens. For whole blood, automated nucleic acid extraction was performed from 1mL ETDA-anticoagulated sample. Results The 95% limit of detection was determined to be 7-10 genomes/reaction by testing remnant DNA eluates spiked with TP gDNA. Assay specificity was evaluated by testing synthetic DNA targets representing non-cultivable targets and residual clinical samples containing DNA from 114 clinically relevant microbes, including non-TP spirochetes, bacteria, fungi, and viruses. TP-LAMP detected 100% of whole blood samples spiked with 120-125 organisms/mL; 88% of samples with 85 organisms/mL; and 80% of samples spiked with 60 organisms/mL. From 09/2022 - 07/2023 TP-LAMP had 110 results in non-blood samples and the mean TAT was 4.7 days. Orthogonal diagnostic results were available for a subset of 34 samples. In this group the assay was 100% sensitive and specific with 7 positive tests, comprising 2 cases of primary and 5 cases of secondary syphilis. Three tissue specimens with negative TP-LAMP results had false-positive immunohistochemical TP stains due to cross-reactivity with commensal treponemes. Conclusion This assay represents a novel, clinically available method for sensitive and specific syphilis diagnosis. Additionally, the assay can resolve ambiguous findings in tissue. Applications in blood may aid in diagnosis of early or latent disease and resolution of unclear serologic test results. Disclosures Stephen J. Salipante, M.D. Ph.D., Anavasi: Grant/Research Support Joshua Lieberman, MD, PhD, Anavasi: Grant/Research Support

Given the resurgence of syphilis, research endeavors to improve current assays for serological diagnosis and management of this disease are a priority. A proteome-scale platform for high-throughput profiling of the humoral response to Treponema pallidum (T. T. pallidum) ) proteins during infection could identify antigens suitable to ameliorate the performance and capabilities of treponemal tests for syphilis. Additionally, because infection-induced immunity is partially protective, profiling the response to T. pallidum outer membrane proteins (OMPs) could help select vaccine candidates. Therefore, we developed a pan-proteome array (PPA) based on the Nichols and SS14 strain complete proteomes and used it to define the immunoglobulin M (IgM) and IgG humoral response to T. pallidum proteins in sera collected longitudinally from long-term infected rabbits and from rabbits that were infected, treated, and re-infected. We identified antigens that could facilitate early diagnosis and immunity to a core set of OMP that could explain protection upon reinfection.

Respiratory syncytial virus (RSV) causes lower respiratory tract infections with significant morbidity and mortality at the extremes of age. Vaccines based on the viral fusion protein are approved for adults over 60, but infant protection relies on passive immunity via antibody transfer or maternal vaccination. An infant vaccine that rapidly elicits protective antibodies would fulfill a critical unmet need. Antibodies arising from the VH3-21/VL1-40 gene pairing can neutralize RSV without the need for affinity maturation, making them attractive to target through vaccination. Here, we develop an anti-idiotypic monoclonal antibody (ai-mAb) immunogen that is specific for unmutated VH3-21/VL1-40 B cell receptors (BCRs). The ai-mAb efficiently engages B cells with bona fide target BCRs and does not activate off-target non-neutralizing B cells, unlike recombinant pre-fusion (preF) protein used in current RSV vaccines. These results establish proof of concept for using an ai-mAb-derived vaccine to target B cells hardwired to produce RSV-neutralizing antibodies.

ABSTRACT Background Given the resurgence of syphilis, research endeavors to improve current assays for serological diagnosis and management of this disease are a priority. A proteome-scale platform for high-throughput profiling of the humoral response to Treponema pallidum ( T. pallidum ) proteins during infection could identify antigens suitable to ameliorate the performance and capabilities of treponemal tests (TTs), which may require weeks to become positive following infection, cannot distinguish between active and previously treated infections, or assess treatment response. Additionally, because infection-induced immunity is partially protective, profiling the response to T. pallidum outer membrane proteins (OMPs) could help select vaccine candidates. Methods We developed a pan-proteome array (PPA) based on the Nichols and SS14 strain complete proteomes and used it to define the IgM and IgG humoral response to 1,009 T. pallidum proteins in sera collected longitudinally from long-term infected rabbits, and from rabbits that were infected, treated, and re-infected. Findings Approximately a third of the pathogen’s proteome was recognized in infected animals, with a marked IgG response detectable between day-10 and day-20 post-infection. We found early, gradual, and late IgG kinetic profiles, strain-dependent differences in humoral reactivity, and post-treatment fluctuation in reactivity for several antigens. Very few antigens elicited an IgM response. Several OMPs were significantly and differentially recognized, but few elicited a robust response. Interpretation The PPA allowed the identification of antigens that could facilitate early diagnosis and of a core set of OMP that could explain protection upon re-infection. No antigen appeared suitable to monitor treatment response. Funding NIH SBIR-R43AI149804 RESEARCH IN CONTEXT Evidence before this study In April 2024, we searched the PubMed database for articles on preclinical studies using high throughput proteome arrays containing at least 10% of the predicted T. pallidum proteome that aimed at identifying antibody reactivity to T. pallidum antigens during experimental syphilis infection. We could retrieve only one manuscript. In this work, an array containing the T. pallidum partial proteome as annotated in the first sequenced Nichols strain genome (GCA_000008605.1) in 1998 was assembled using recombinant antigens expressed in Escherichia coli ( E. coli ). The resulting array was probed using pooled sera from three rabbits infected with the Nichols stain of T. pallidum , attained from infected animals at five time points following intratesticular infection. The small number of reactive antigens (n = 106) identified in this early study was likely to be an incomplete set of all antigens recognized during infection because not all the predicted targets in the T. pallidum proteome were successfully expressed and tested. In retrospect, additional limitations of the study included an initial suboptimal annotation of the Nichols genome used to define the pathogen’s proteome, which has now changed with the availability of a re-sequenced Nichols strain genome devoid of sequencing errors that affected the initial annotation process, and the refinement of bioinformatic pipelines for the identification of open reading frames (ORFs). Furthermore (as acknowledged by the authors), the possible presence of amplification errors in their expression clones might have affected the sequence of some protein targets and antibody binding to the targets. As a result, some of the T. pallidum antigens known to elicit a robust humoral response during experimental infection were not detected in this antigenic screen. Lastly, employing only the Nichols strain in this early study did not consider that a significant portion of the circulating syphilis strains belong to the SS14 clade of T. pallidum . Added value of this study This novel PPA, combined with a more robust experiential design than ever reported, allowed us to overcome most of the limitations associated with the study mentioned above, as we were able to a) use the most recent annotations for the selected T. pallidum strains based on accurate genome sequences, b) print the pathogen’s virtually complete proteome in the study array, c) analyze individual sera to account for rabbit-to-rabbit variability in the humoral response to infection rather than pooled sera, d) detect both IgM and IgG over 10 or 20 timepoints, depending on the experimental design, e) obtain information on how the humoral response evolved upon treatment and re-infection and, finally, f) evaluate all of the above in animals infected with two T. pallidum strains whose genetic background is representative of the two currently circulating clades of the syphilis agent. Implications of all the available evidence Our study provides new and more comprehensive data on how humoral immunity for two classes of antibodies develops during infection and how it evolves in response to treatment and re-infection. The analysis of sera collected at tightly spaced time points post-inoculation and for an extensive period post-infection provides a wealth of information to improve the diagnostic performance of existing tests detecting treponemal antigens. The analysis of differential immunity specific to the pathogen’s putative OMPs provides a rationale for vaccine candidate selection.

ABSTRACT Syphilis is a multistage chronic sexually transmitted infection caused by the spirochete Treponema pallidum subsp. pallidum (T. pallidum). Today, syphilis is still endemic in low- and middle-income nations and has become resurgent in many high-income countries. Infection can lead to severe sequelae in untreated patients, while vertical transmission of T. pallidum is associated with high stillbirth rates and neonatal death. The combination of non-treponemal and treponemal tests allows syphilis serodiagnosis and monitoring of treatment response, albeit not without limitations. In this work, we developed a minimal array for multiple parallel detection of antibodies specific to the immunodominant T. pallidum antigens Tp0435 and Tp0574 and 14 additional proteins known to be immunogenic during infection but only partially or not at all evaluated for their diagnostic potential. To assess whether reactivity to these antigens could improve early syphilis diagnosis, serve as biomarkers for disease staging, and monitor response to treatment, we tested the array using 217 serum specimens longitudinally collected pre- and post-treatment from 120 syphilis patients. Results showed significantly different reactivity to a subset of antigens in pre-treatment sera (baseline) when covariates such as syphilis stage, syphilis history, and HIV status were factored in. Furthermore, reactivity to several antigens significantly decreased in post-treatment sera compared to baseline. Although studies with larger sample panels will be needed to validate these findings, this study supports the screening of T. pallidum proteomic arrays to identify antigens that could serve as predictors of response and antigens that could help with early syphilis diagnosis. IMPORTANCE This manuscript explores the host humoral response to selected antigens of the syphilis agent during infection to evaluate their potential use as diagnostic tests and markers for treatment.

Sequencing of most Treponema pallidum genomes excludes repeat regions in tp0470 and the tp0433 gene, encoding the acidic repeat protein (arp). As a first step to understanding the evolution and function of these genes and the proteins they encode, we developed a protocol to nanopore sequence tp0470 and arp genes from 212 clinical samples collected from ten countries on six continents. Both tp0470 and arp repeat structures recapitulate the whole genome phylogeny, with subclade-specific patterns emerging. The number of tp0470 repeats is on average appears to be higher in Nichols-like clade strains than in SS14-like clade strains. Consistent with previous studies, we found that 14-repeat arp sequences predominate across both major clades, but the combination and order of repeat type varies among subclades, with many arp sequence variants limited to a single subclade. Although strains that were closely related by whole genome sequencing frequently had the same arp repeat length, this was not always the case. Structural modeling of TP0470 suggested that the eight residue repeats form an extended α-helix, predicted to be periplasmic. Modeling of the ARP revealed a C-terminal sporulation-related repeat (SPOR) domain, predicted to bind denuded peptidoglycan, with repeat regions possibly incorporated into a highly charged β-sheet. Outside of the repeats, all TP0470 and ARP amino acid sequences were identical. Together, our data, along with functional considerations, suggests that both TP0470 and ARP proteins may be involved in T. pallidum cell envelope remodeling and homeostasis, with their highly plastic repeat regions playing as-yet-undetermined roles.

Immune evasion by Treponema pallidum subspecies pallidum ( T. pallidum ) has been attributed to antigenic variation of its putative outer-membrane protein TprK. In TprK, amino acid diversity is confined to seven variable (V) regions, and generation of sequence diversity within the V regions occurs via a non-reciprocal segmental gene conversion mechanism where donor cassettes recombine into the tprK expression site. Although previous studies have shown the significant role of immune selection in driving accumulation of TprK variants, the contribution of baseline gene conversion activity to variant diversity is less clear. Here, combining longitudinal tprK deep sequencing of near clonal Chicago C from immunocompetent and immunosuppressed rabbits along with the newly developed in vitro cultivation system for T. pallidum , we directly characterized TprK alleles in the presence and absence of immune selection. Our data confirm significantly greater sequence diversity over time within the V6 region during syphilis infection in immunocompetent rabbits compared to immunosuppressed rabbits, consistent with previous studies on the role of TprK in evasion of the host immune response. Compared to strains grown in immunocompetent rabbits, strains passaged in vitro displayed low level changes in allele frequencies of TprK variable region sequences similar to that of strains passaged in immunosuppressed rabbits. Notably, we found significantly increased rates of V6 allele generation relative to other variable regions in in vitro cultivated T, pallidum strains, illustrating that the diversity within these hypervariable regions occurs in the complete absence of immune selection. Together, our results demonstrate antigenic variation in T. pallidum can be studied in vitro and occurs even in the complete absence of immune pressure, allowing the T. pallidum population to continuously evade the immune system of the infected host. Author Summary Syphilis continues to be a disease of global and public health concern, even though the infection can be easily diagnosed and effectively treated with penicillin. Although infected individuals often develop a strong immunity to the pathogen, repeated infection with the syphilis agent, Treponema pallidum subspecies pallidum ( T. pallidum ), is possible. Several studies point at antigenic variation of the T. pallidum TprK protein as the mechanism responsible for evasion of the immunity that develops during infection, pathogen persistence, and re-infection. Past studies have highlighted the importance of immune clearance of dominant variants that, in turn, allows less represented variants to emerge. The contribution of an immunity-independent baseline generation of variability in the tprK gene is less clear. Here, we used deep sequencing to profile tprK variants using a laboratory-isolated T. pallidum strain nearly isogenic for tprK that was propagated over time in vitro , where no immune pressure is exerted on the pathogen, as well as in samples obtained from immunosuppressed and immunocompetent rabbits infected with the same strain. We confirmed that tprK accumulates significantly more diversity under immune pressure, and demonstrated a low but discernible basal rate of gene conversion in complete absence of immune pressure.

Immune evasion by Treponema pallidum subspecies pallidum (T. pallidum) has been attributed to antigenic variation of its putative outer-membrane protein TprK. In TprK, amino acid diversity is confined to seven variable (V) regions, and generation of sequence diversity within the V regions occurs via a non-reciprocal segmental gene conversion mechanism where donor cassettes recombine into the tprK expression site. Although previous studies have shown the significant role of immune selection in driving accumulation of TprK variants, the contribution of baseline gene conversion activity to variant diversity is less clear. Here, combining longitudinal tprK deep sequencing of near clonal Chicago C from immunocompetent and immunosuppressed rabbits along with the newly developed in vitro cultivation system for T. pallidum, we directly characterized TprK alleles in the presence and absence of immune selection. Our data confirm significantly greater sequence diversity over time within the V6 region during syphilis infection in immunocompetent rabbits compared to immunosuppressed rabbits, consistent with previous studies on the role of TprK in evasion of the host immune response. Compared to strains grown in immunocompetent rabbits, strains passaged in vitro displayed low level changes in allele frequencies of TprK variable region sequences similar to that of strains passaged in immunosuppressed rabbits. Notably, we found significantly increased rates of V6 allele generation relative to other variable regions in in vitro cultivated T, pallidum strains, illustrating that the diversity within these hypervariable regions occurs in the complete absence of immune selection. Together, our results demonstrate antigenic variation in T. pallidum can be studied in vitro and occurs even in the complete absence of immune pressure, allowing the T. pallidum population to continuously evade the immune system of the infected host.

Patients with complicated inflammatory bowel disease commonly undergo repeated surgical procedures, often against a background of chronic opiate use. We describe a case in which a postoperative attempt to withdraw opiate analgesia on two separate occasions led to a clinical syndrome strongly suggestive of intestinal obstruction, the signs and symptoms of which settled rapidly on re-introduction of opiates. Small bowel contrast studies indicated a level of obstruction which not only fluctuated, but occurred at an unusual site for mechanical obstruction. in patients with a history of long-standing opiate use, postoperative opiate withdrawal can cause a significant, functional bowel disorder and should be borne in mind in the differential diagnosis of postoperative intestinal obstruction. Patients can be treated effectively with clonidine.

Background: Penicillin G, the current standard treatment for syphilis, has important drawbacks, but virtually no preclinical or clinical studies have been performed to identify viable alternatives. We tested, both in vitro and in vivo, three marketed antibiotics with adequate pharmacological properties to treat syphilis. Methods: We used an in vitro culturing system of T. pallidum to perform drug susceptibility testing and applied quantitative PCR targeting the tp0574 gene to measure bacterial growth. To confirm in vivo efficacy, fifteen rabbits were infected intradermally with T. pallidum at eight sites each and randomly allocated to an experimental treatment (linezolid, moxifloxacin, clofazimine) or a control arm (benzathine penicillin G [BPG], untreated). The primary outcome was treatment efficacy defined as the time to lesion healing measured from the date of treatment start. Secondary outcomes were absence of treponemes or treponemal mRNA in injection sites, absence of seroconversion, and cerebrospinal fluid (CSF) abnormalities and negative rabbit infectivity tests (RIT). Findings: Linezolid showed in vitro bactericidal activity at concentrations of 0.5 µg/mL or higher. When administered orally to experimentally infected rabbits, it induced healing of early lesions at a time similar to BPG (hazard ratio 3.84; 95% CI 2.05–7.17; p < 0.0001 compared to untreated controls). In linezolid-treated animals, dark-field microscopy and qPCR assessment showed no presence of treponemes after day 3 post-treatment start, serologic test did not convert to positive, CSF had no abnormalities, and RIT was negative. Moxifloxacin and clofazimine failed to inhibit bacterial growth in vitro and could not cure the infection in the rabbit model. Interpretation: Linezolid, a low-cost oxazolidinone, has in vitro and in vivo activity against T. pallidum, with efficacy similar to BPG in treating treponemal lesions in the animal model. Our findings warrant further research to assess the efficacy of linezolid as an alternative to penicillin G to treat syphilis in human clinical trials. Funding: European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (Grant agreement No. 850450).

Background Penicillin G (PG), the current standard for syphilis treatment, has important drawbacks that hamper public health efforts to curtail the spread of this infection, particularly in low- and middle-income countries where syphilis is still endemic. Methods To identify viable alternatives to PG, we tested—both in vitro and in vivo—three marketed antibiotics with adequate pharmacokinetic and pharmacodynamic properties to treat syphilis. Results We found that linezolid, an oxazolidinone overcoming most of the current drawbacks of PG, showed in vitro bactericidal activity at concentrations of 0.5 µg/mL or higher. When administered orally to experimentally infected rabbits, it induced healing of early lesions at a similar time of PG and effectively treated the infection. The hazard ratio for healing was 3.84 (95% CI 2.05 – 7.17; p < 0.0001) compared to untreated controls. Conclusions Our findings warrant further research to assess the efficacy of linezolid as an alternative to PG to treat early syphilis in clinical settings.

In spite of its immutable susceptibility to penicillin,Treponema pallidum(T.pallidum) subsp.pallidumcontinues to cause millions of cases of syphilis each year worldwide, resulting in significant morbidity and mortality and underscoring the urgency of developing an effective vaccine to curtail the spread of the infection. Several technical challenges, including absence of anin vitroculture system until very recently, have hampered efforts to catalog the diversity of strains collected worldwide. Here, we provide near-complete genomes from 196T.pallidumstrains–including 191T.pallidumsubsp.pallidum–sequenced directly from patient samples collected from 8 countries and 6 continents. Maximum likelihood phylogeny revealed that samples from most sites were predominantly SS14 clade. However, 99% (84/85) of the samples from Madagascar formed two of the five distinct Nichols subclades. Although recombination was uncommon in the evolution of modern circulating strains, we found multiple putative recombination events betweenT.pallidumsubsp.pallidumand subsp.endemicum, shaping the genomes of several subclades. Temporal analysis dated the most recent common ancestor of Nichols and SS14 clades to 1717 (95% HPD: 1543–1869), in agreement with other recent studies. Rates of SNP accumulation varied significantly among subclades, particularly among different Nichols subclades, and was associated in the Nichols A subclade with a C394F substitution in TP0380, a ERCC3-like DNA repair helicase. Our data highlight the role played by variation in genes encoding putative surface-exposed outer membrane proteins in defining separate lineages, and provide a critical resource for the design of broadly protective syphilis vaccines targeting surface antigens.

Background The syphilis epidemic continues to cause substantial morbidity and mortality worldwide, particularly in low- and middle-income countries, despite several recent disease control initiatives. Though our understanding of the pathogenesis of this disease and the biology of the syphilis agent, Treponema pallidum subsp. pallidum has improved over the last two decades, further research is necessary to improve clinical diagnosis and disease management protocols. Additionally, such research efforts could contribute to the identification of possible targets for the development of an effective vaccine to stem syphilis spread. Methods This study will recruit two cohorts of participants with active syphilis infection, one with de novo infection, one with repeat infection. Whole blood specimens will be collected from each study participant at baseline, 4, 12, 24, 36, and 48 weeks, to track specific markers of their immunological response, as well as to compare humoral reactivity to Treponema pallidum antigens between the two groups. Additionally, we will use serum specimens to look for unique cytokine patterns in participants with early syphilis. Oral and blood samples, as well as samples from any syphilitic lesions present, will also be collected to sequence any Treponema pallidum DNA found. Discussion By furthering our understanding of syphilis pathogenesis and human host immune response to Treponema pallidum , we will provide important data that will help in development of new point-of-care tests that could better identify active infection, leading to improved syphilis diagnosis and management. Findings could also contribute to vaccine development efforts.

BACKGROUND:Gallium is a semi-metallic element known since the 1930s to have antimicrobial activity. This activity stems primarily from gallium's ability to mimic trivalent iron and disrupt specific Fe(III)-dependent pathways, particularly DNA synthesis (due to inhibition of ribonucleotide reductase). Because of its novel mechanism of action, gallium is currently being investigated as a new antibacterial agent, particularly in light of the increasing resistance of many pathogenic bacteria to existing antibiotics. Gallium maltolate (GaM) is being developed as an orally and topically administrable form of gallium. Yaws is a neglected tropical disease affecting mainly the skin and skeletal system of children in underprivileged settings. It is currently the object of a WHO-promoted eradication campaign using mass administration of the macrolide azithromycin, an antibiotic to which the yaws agent Treponema pallidum subsp. pertenue has slowly begun to develop genetic resistance.METHODS:Because yaws transmission is mainly due to direct skin contact with an infectious skin lesion, we evaluated the treponemicidal activity of GaM applied topically to skin lesions in a rabbit model of yaws. Treatment efficacy was evaluated by measuring lesion diameter, treponemal burden in lesion aspirates as determined by dark field microscopy and amplification of treponemal RNA, serology, and immunohistochemistry of biopsied tissue samples.RESULTS:Our results show that topical GaM was effective in reducing treponemal burden in yaws experimental lesions, particularly when applied at the first sign of lesion appearance but, as expected, did not prevent pathogen dissemination.CONCLUSION:Early administration of GaM to yaws lesions could reduce the infectivity of the lesions and thus yaws transmission, potentially contributing to current and future yaws control campaigns.

In silico analyses of Treponema pallidum subsp. pallidum genomes and predicted proteomes to search for homologs of known bacterial outer membrane proteins (OMPs) led to the identification of tp0126 as a gene encoding a putative member of the OmpW family of porins/virulence factors. Our previous investigations on the role of Tp0126 in T. pallidum biology and syphilis pathogenesis showed that Tp0126 is fully conserved among T. pallidum strains and that transcription of tp0126 is driven by σ70 These initial results pointed to a housekeeping function for this protein. We also demonstrated that a guanosine homopolymer of various lengths located between the -10 and -35 consensus sequences in the tp0126 promoter modulates transcription consistently with phase variation, a mechanism that we also previously described for other T. pallidum genes encoding putative OMPs/virulence factors and that is often employed as a strategy for immune evasion. Circular dichroism spectra of recombinant Tp0126 also supported its structural homology with OmpW. Here we further investigated the humoral and cellular responses to Tp0126 during experimental and natural syphilis and the ability of Tp0126 to confer protection against syphilis in immunized rabbits. B-cell epitope mapping showed that compared to sera from experimentally infected animals, immunizations enhanced humoral immunity to sequences located in the putative Tp0126 surface-exposed loops, while phagocytosis assays showed that postimmunization sera opsonized T. pallidum Despite such promising results, no significant protection was seen following infectious challenge in immunized animals versus controls. Functional redundancy and phase variation might explain the lack of effectiveness of this vaccine candidate and/or design.

BACKGROUND:Syphilis is resurgent in many developed countries and still prevalent in developing nations. Current and future control campaigns would benefit from the development of a vaccine, but although promising vaccine candidates were identified among the putative surface-exposed integral outer membrane proteins of the syphilis spirochete, immunization experiments in the rabbit model using recombinant antigens have failed to fully protect animals upon infectious challenge. We speculated that such recombinant immunogens, purified under denaturing conditions from Escherichia coli prior to immunization might not necessarily harbor their original structure, and hypothesized that enhanced protection would result from performing similar immunization/challenge experiments with native antigens. METHODS:To test our hypothesis, we engineered non-infectious Borrelia burgdorferi strains to express the tp0897 (tprK) and tp0435 genes of Treponema pallidum subsp. pallidum and immunized two groups of rabbits by injecting recombinant strains intramuscularly with no adjuvant. TprK is a putative integral outer membrane protein of the syphilis agent, while tp0435 encodes the highly immunogenic T. pallidum 17-kDa lipoprotein, a periplasmic antigen that was also shown on the pathogen surface. Following development of a specific host immune response to these antigens as the result of immunization, animals were challenged by intradermal inoculation of T. pallidum. Cutaneous lesion development was monitored and treponemal burden within lesions were assessed by dark-field microscopy and RT-qPCR, in comparison to control rabbits. RESULTS:Partial protection was observed in rabbits immunized with B. burgdorferi expressing TprK while immunity to Tp0435 was not protective. Analysis of the humoral response to TprK antigen suggested reactivity to conformational epitopes. CONCLUSIONS:Immunization with native antigens might not be sufficient to obtain complete protection to infection. Nonetheless we showed that non-infectious B. burgdorferi can be an effective carrier to deliver and elicit a specific host response to T. pallidum antigens to assess the efficacy of syphilis vaccine candidates.

Background The bacterium that causes syphilis, Treponema pallidum subsp. pallidum (Tp), elicits cellular and humoral immune responses to numerous antigens during infection. We have identified three recombinant peptide antigens that, when used separately for immunization, show promise in the attenuation of chancre development or dissemination to distant tissues. Here, we report protection induced by a three-antigen cocktail emulsified in either of two custom adjuvants containing Natural or Synthetic TLR4 agonists + a natural Mincle agonist. Methods Three purified recombinant peptides [TprK (aa37-273), Tp0751 (aa24-237), and Tpr Subfamily I (23-351)] were emulsified in either adjuvant and used to immunize groups of 8 rabbits. The immunized rabbits and 8 Unimmunized controls were challenged intradermally with 10^5Tp/site at 10 sites. Lesion development was recorded daily. Treponemal burden was measured by darkfield (DF) microscopy and qPCR of lesion aspirates, and dissemination to distant tissues was evaluated by rabbit infectivity test (RIT). Results Compared to Unimmunized, treponemal burden by DF in lesion aspirates at Day 19 was lower in both Natural (P=0.001) and Synthetic (P=0.004) groups; by qPCR, treponemal burden was lower in the Natural group (P=0.008). At Days 19 and 30, the proportion of lesions ulcerating was lower in the Natural group, compared to Unimmunized (P=0.0001 [d.19] and P=0.0002, [d.30]). At day 30, the proportion of lesions ulcerating in the Natural group was lower than in the Synthetic (P=0.04) group. Mean lesion volume was smaller in immunized groups versus Unimmunized on days 10–25. RIT indicated the lowest number of disseminated Tp in rabbit tissues from the Natural group, followed by the Synthetic group, then the Unimmunized group (P=0.0247). Conclusion Immunization with the three-antigen cocktail significantly attenuates syphilis infection: enhancing Tp clearance, promoting lesion healing, and reducing dissemination. In rabbits, Natural adjuvant was more effective than Synthetic adjuvant in inducing protective immunity. Disclosure No significant relationships.