We have identified a family of genes that code for targets for opsonic antibody and protective immunity in T. pallidum subspecies pallidum using two different approaches, subtraction hybridization and differential immunologic screening of a T. pallidum genomic library. Both approaches led to the identification of a polymorphic multicopy gene family with predicted amino acid homology to the major sheath protein of Treponema denticola. One of the members of this gene family, tpr K, codes for a protein that is predicted to have a cleavable signal peptide and be located in the outer membrane of the bacterium. Reverse transcription polymerase chain reaction analysis of T. pallidum reveals that Tpr K is preferentially transcribed in the Nichols strain of T. pallidum. Antibodies directed to purified recombinant variable domain of Tpr K can opsonize T. pallidum, Nichols strain, for phagocytosis, supporting the hypothesis that this portion of the protein is exposed at the surface of the treponeme. Immunization of rabbits with the purified recombinant variable domain of Tpr K provides significant protection against infection with the Nichols strain of T. pallidum. This gene family is hypothesized to be central to pathogenesis and immunity during syphilis infection.

By means of a differential screening technique, a 92-kDa antigen, designated Tp92, was identified from Treponema pallidum subspecies pallidum. This protein is similar in sequence to the protective surface antigens D15 from Haemophilus influenzae and Oma87 from Pasteurella multocida. Amino acid sequence analyses revealed a cleavable N-terminal signal sequence and predicted the outer membrane location for Tp92. In support of this, antiserum raised against recombinant Tp92 promotes opsonization and phagocytosis of T. pallidum by rabbit macrophages, and anti-Tp92 reactivity is absent from washed treponemal preparations presumed to be lacking outer membranes. The Tp92 amino acid sequence is 95.5%-100% conserved among 11 strains representing 4 pathogenic treponemes, and immunization with recombinant Tp92 partially protected rabbits from subsequent T. pallidum challenge. These results demonstrate that Tp92 is an invariant, immunoprotective antigen that may be present on the surface of T. pallidum and may represent a potential vaccine candidate for syphilis.

SUMMARY Definitive identification of Treponema pallidum (Tp) rare outer membrane proteins (OMPs) has long eluded researchers. TP0326, the sole protein in Tp with sequence homology to a Gram-negative OMP, belongs to the BamA family of proteins essential for OM biogenesis. Structural modeling predicted that five polypeptide transport-associated (POTRA) domains comprise the N-terminus of TP0326, while the C-terminus forms an 18-stranded amphipathic β-barrel. Circular dichroism, heat-modifiability by SDS-PAGE, Triton X-114 phase partitioning and liposome incorporation supported these topological predictions and confirmed that the β-barrel is responsible for the native protein's amphiphilicity. Expression analyses revealed that native TP0326 is expressed at low abundance, while a protease-surface accessibility assay confirmed surface exposure. Size-exclusion chromatography and blue native polyacrylamide gel electrophoresis revealed a modular Bam complex in Tp considerably larger than that of E. coli. Non-orthologous ancillary factors and self-association of TP0326 via its β-barrel may both contribute to the Bam complex. Tp-infected rabbits mount a vigorous antibody response to both POTRA and β-barrel portions of TP0326, whereas humans with secondary syphilis respond predominantly to POTRA. The syphilis spirochete appears to have devised a stratagem for harnessing the Bam pathway while satisfying its need to limit surface antigenicity.

In 2014, the World Health Organization, the US National Institutes of Health, and global technical partners published a comprehensive roadmap for development of new vaccines against sexually transmitted infections (STIs). Since its publication, progress has been made in several roadmap activities: obtaining better epidemiologic data to establish the public health rationale for STI vaccines, modeling the theoretical impact of future vaccines, advancing basic science research, defining preferred product characteristics for first-generation vaccines, and encouraging investment in STI vaccine development. This article reviews these overarching roadmap activities, provides updates on research and development of individual vaccines against herpes simplex virus, Chlamydia trachomatis, Neisseria gonorrhoeae, and Treponema pallidum, and discusses important next steps to advance the global roadmap for STI vaccine development.

Treponema pallidum, the causative agent of syphilis, is a highly invasive pathogenic spirochete capable of attaching to host cells, invading the tissue barrier, and undergoing rapid widespread dissemination via the circulatory system. The T. pallidum adhesin Tp0751 was previously shown to bind laminin, the most abundant component of the basement membrane, suggesting a role for this adhesin in host tissue colonization and bacterial dissemination. We hypothesized that similar to that of other invasive pathogens, the interaction of T. pallidum with host coagulation proteins, such as fibrinogen, may also be crucial for dissemination via the circulatory system. To test this prediction, we used enzyme-linked immunosorbent assay (ELISA) methodology to demonstrate specific binding of soluble recombinant Tp0751 to human fibrinogen. Click-chemistry-based palmitoylation profiling of heterologously expressed Tp0751 confirmed the presence of a lipid attachment site within this adhesin. Analysis of the Tp0751 primary sequence revealed the presence of a C-terminal putative HEXXH metalloprotease motif, and in vitro degradation assays confirmed that recombinant Tp0751 purified from both insect and Escherichia coli expression systems degrades human fibrinogen and laminin. The proteolytic activity of Tp0751 was abolished by the presence of the metalloprotease inhibitor 1,10-phenanthroline. Further, inductively coupled plasma-mass spectrometry showed that Tp0751 binds zinc and calcium. Collectively, these results indicate that Tp0751 is a zinc-dependent, membrane-associated protease that exhibits metalloprotease-like characteristics. However, site-directed mutagenesis of the HEXXH motif to HQXXH did not abolish the proteolytic activity of Tp0751, indicating that further mutagenesis studies are required to elucidate the critical active site residues associated with this protein. This study represents the first published description of a T. pallidum protease capable of degrading host components and thus provides novel insight into the mechanism of T. pallidum dissemination.