Dataset Information

Immunogenicity and protective efficacy against Treponema pallidum in New Zealand rabbits immunized with plasmid DNA encoding flagellin.

ABSTRACT: Plasmid DNA encoding flagellin FlaB3 was used as a vaccination candidate for the evaluation of immunogenicity and protection against Treponema pallidum subsp. pallidum dissemination. First, intramuscular injection of the flagellin encoded by the plasmid DNA into New Zealand rabbits elicited both humoral and cellular immune responses. Total IgG production increased in response to flagellin. In addition, serum IFN-? secretion and CD8+ cells were substantially greater in the rabbits immunized with the plasmid encoding flagellin FlaB3 than those in the rabbits immunized with recombinant flagellin. The flagellin encoded by the plasmid DNA induced significant upregulation of serum IL-6 and IL-8 compared to that of the control rabbits. Subsequently, intradermal challenge of the vaccinated New Zealand rabbits with 1?×?107T. pallidum resulted in a significant reduction of the bacterial organ burden in the blood, liver, spleen, and testicles in the flagellin plasmid DNA-vaccinated rabbits. Furthermore, the histopathological analysis demonstrated that the rabbits immunized with the plasmid DNA-encoded flagellin (FlaB3) showed better immune protection. These findings provide evidence that plasmid DNA-encoded flagellin (FlaB3) may be useful as a potential immunization route for future development of a vaccine to inhibit T. pallidum dissemination in related animals.

SUBMITTER: Zheng K

PROVIDER: S-EPMC6220273 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Immunogenicity and protective efficacy against Treponema pallidum in New Zealand rabbits immunized with plasmid DNA encoding flagellin.

Zheng Kang K Xu Man M Xiao Yongjian Y Luo Haodang H Xie Yafeng Y Yu Jian J Tan Manyi M Li Yumeng Y Zhao Feijun F Zeng Tiebing T Wu Yimou Y

Emerging microbes & infections 20181107 1

Plasmid DNA encoding flagellin FlaB3 was used as a vaccination candidate for the evaluation of immunogenicity and protection against Treponema pallidum subsp. pallidum dissemination. First, intramuscular injection of the flagellin encoded by the plasmid DNA into New Zealand rabbits elicited both humoral and cellular immune responses. Total IgG production increased in response to flagellin. In addition, serum IFN-γ secretion and CD8+ cells were substantially greater in the rabbits immunized with ...[more]

PMID: 30405111

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Project description:Despite more than a century of research, genetic manipulation of Treponema pallidum subsp. pallidum (T. pallidum), the causative agent of syphilis, has not been successful. The lack of genetic engineering tools has severely limited understanding of the mechanisms behind T. pallidum success as a pathogen. A recently described method for in vitro cultivation of T. pallidum, however, has made it possible to experiment with transformation and selection protocols in this pathogen. Here, we describe an approach that successfully replaced the tprA (tp0009) pseudogene in the SS14 T. pallidum strain with a kanamycin resistance (kanR) cassette. A suicide vector was constructed using the pUC57 plasmid backbone. In the vector, the kanR gene was cloned downstream of the tp0574 gene promoter. The tp0574prom-kanR cassette was then placed between two 1-kbp homology arms identical to the sequences upstream and downstream of the tprA pseudogene. To induce homologous recombination and integration of the kanR cassette into the T. pallidum chromosome, in vitro-cultured SS14 strain spirochetes were exposed to the engineered vector in a CaCl2-based transformation buffer and let recover for 24 hours before adding kanamycin-containing selective media. Integration of the kanR cassette was demonstrated by qualitative PCR, droplet digital PCR (ddPCR), and whole-genome sequencing (WGS) of transformed treponemes propagated in vitro and/or in vivo. ddPCR analysis of RNA and mass spectrometry confirmed expression of the kanR message and protein in treponemes propagated in vitro. Moreover, tprA knockout (tprAko-SS14) treponemes grew in kanamycin concentrations that were 64 times higher than the MIC for the wild-type SS14 (wt-SS14) strain and in infected rabbits treated with kanamycin. We demonstrated that genetic manipulation of T. pallidum is attainable. This discovery will allow the application of functional genetics techniques to study syphilis pathogenesis and improve syphilis vaccine development.

Project description:Immune evasion and disease progression of Treponema pallidum subsp. pallidum are associated with sequence diversity in the hypervariable outer membrane protein TprK. Previous attempts to study variation within TprK have sequenced at depths insufficient to fully appreciate the hypervariable nature of the protein, failed to establish linkage between the protein's seven variable regions, or were conducted on isolates passed through rabbits. As a consequence, a complete profile of tprK during infection in the human host is still lacking. Furthermore, prior studies examining how T. pallidum subsp. pallidum uses its repertoire of genomic donor sites to generate diversity within the variable regions of the tprK have yielded a partial understanding of this process due to the limited number of tprK alleles examined. In this study, we used short- and long-read deep sequencing to directly characterize full-length tprK alleles from T. pallidum subsp. pallidum collected from early lesions of patients attending two sexually transmitted infection clinics in Italy. We demonstrate that strains collected from cases of secondary syphilis contain significantly more unique variable region sequences and full-length TprK sequences than those from cases of primary syphilis. Our data, combined with recent data available on Chinese T. pallidum subsp. pallidum specimens, show the near-complete absence of overlap in TprK sequences among the 41 specimens profiled to date. We further estimate that the potential antigenic variability carried by TprK rivals that of current estimates of the human adaptive immune system. These data underscore the immunoevasive ability of TprK that allows T. pallidum subsp. pallidum to establish lifelong infection.IMPORTANCE Syphilis continues to be a significant public health issue in both low- and high-income countries, including the United States where the rate of syphilis infection has increased over the past 5 years. Treponema pallidum subsp. pallidum, the causative agent of syphilis, carries the outer membrane protein TprK that undergoes segmental gene conversion to constantly create new sequences. We performed full-length deep sequencing of TprK to examine TprK diversity in clinical T. pallidum subsp. pallidum strains. We then combined our results with data from all samples for which TprK deep sequencing results were available. We found almost no overlap in TprK sequences between different patients. Moreover, our data allowed us to estimate the total number of TprK variants that T. pallidum subsp. pallidum can potentially generate. Our results support how the T. pallidum subsp. pallidum TprK antigenic variation system is an equal adversary of the human immune system leading to pathogen persistence in the host.

Dataset Information

Immunogenicity and protective efficacy against Treponema pallidum in New Zealand rabbits immunized with plasmid DNA encoding flagellin.

Publications

Immunogenicity and protective efficacy against Treponema pallidum in New Zealand rabbits immunized with plasmid DNA encoding flagellin.

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