Non-pathogenic Borrelia burgdorferi expressing Treponema pallidum TprK and Tp0435 antigens as a novel approach to evaluate syphilis vaccine candidates.

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.

Topical treatment with gallium maltolate reduces Treponema pallidum subsp. pertenue burden in primary experimental lesions in a rabbit model of yaws.

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.

Two mutations associated with macrolide resistance in Treponema pallidum: increasing prevalence and correlation with molecular strain type in Seattle, Washington.

BACKGROUND: Although azithromycin promised to be a safe and effective single-dose oral treatment of early syphilis, azithromycin treatment failure has been documented and is associated with mutations in the 23S rDNA of corresponding Treponema pallidum strains. The prevalence of strains harboring these mutations varies throughout the United States and the world. We examined T. pallidum strains circulating in Seattle, Washington, from 2001 to 2010 to determine the prevalence of 2 mutations associated with macrolide resistance and to determine whether these mutations were associated with certain T. pallidum strain types. METHODS: Subjects were enrolled in a separate ongoing study of cerebrospinal fluid abnormalities in patients with syphilis. T. pallidum DNA purified from blood and T. pallidum strains isolated from blood or cerebrospinal fluid were analyzed for two 23S rDNA mutations and for the molecular targets used in an enhanced molecular stain typing system. RESULTS: Nine molecular strain types of T. pallidum were identified in Seattle from 2001 to 2010. Both macrolide resistance mutations were identified in Seattle strains, and the prevalence of resistant T. pallidum exceeded 80% in 2005 and increased through 2010. Resistance mutations were associated with discrete molecular strain types of T. pallidum. CONCLUSIONS: Macrolide-resistant T. pallidum strains are highly prevalent in Seattle, and each mutation is associated with discrete strain types. Macrolides should not be considered for treatment of syphilis in regions where prevalence of the mutations is high. Combining the resistance mutations with molecular strain typing permits a finer analysis of the epidemiology of syphilis in a community.

Antigenic variation in Treponema pallidum: TprK sequence diversity accumulates in response to immune pressure during experimental syphilis.

Pathogens that cause chronic infections often employ antigenic variation to evade the immune response and persist in the host. In Treponema pallidum (T. pallidum), the causative agent of syphilis, the TprK Ag undergoes variation of seven V regions (V1-V7) by nonreciprocal recombination of silent donor cassettes with the tprK expression site. These V regions are the targets of the host humoral immune response during experimental infection. The present study addresses the causal role of the acquired immune response in the selection of TprK variants in two ways: 1) by investigating TprK variants arising in immunocompetent versus immunosuppressed hosts; and 2) by investigating the effect of prior specific immunization on selection of TprK variants during infection. V region diversity, particularly in V6, accumulates more rapidly in immunocompetent rabbits than in pharmacologically immunosuppressed rabbits (treated with weekly injections of methylprednisolone acetate). In a complementary experiment, rabbits preimmunized with V6 region synthetic peptides had more rapid accumulation of V6 variant treponemes than control rabbits. These studies demonstrate that the host immune response selects against specific TprK epitopes expressed on T. pallidum, resulting in immune selection of new TprK variants during infection, confirming a role for antigenic variation in syphilis.