22Immune evasion and disease progression of Treponema pallidum subspecies pallidum 23 are associated with sequence diversity in the hypervariable, putative outer membrane 24 protein TprK. Previous attempts to study variation within TprK have sequenced at 25 depths insufficient to fully appreciate the hypervariable nature of the protein, failed to 26 establish linkage between the protein's 7 variable regions, or were conducted on strains 27 passed through rabbits. As a consequence, a complete profiling of tprK during infection 28 in the human host is still lacking. Furthermore, prior studies examining how T. pallidum 29 uses its repertoire of genomic donor sites to generate diversity within the V regions of 30 the tprK also yielded a partial understanding of this process, due to the limited number 31 of tprK alleles examined. In this study, we used short-and long-read deep sequencing 32 to directly characterize full-length tprK alleles from T. pallidum collected from early 33 lesions of patients attending two STD clinics in Italy. Our data, combined with recent 34 data available on Chinese T. pallidum strains, show the near complete absence of 35 overlap in TprK sequences among the 41 strains profiled to date. Moreover, our data 36 allowed us to redefine the boundaries of tprK V regions, identify 55 donor sites, and 37 estimate the total number of TprK variants that T. pallidum can potentially generate. 38Altogether, our results support how T. pallidum TprK antigenic variation system is an 39 unsurmountable obstacle for the human immune system to naturally achieve infection 40 eradication, and reiterate the importance of this mechanism for pathogen persistence in 41 the host. 42 43 Importance 44 Syphilis continues to be a significant public health issue in both low-and high-income 45 nations, including the United States, where the number of infectious syphilis cases has 46 increased dramatically over the past five years. T. pallidum, the causative agent of 47 that the total baseline junctional diversity of full-length TprK rivals that of current 53 estimates of the human adaptive immune system. These data underscore the 54 immunoevasive ability of TprK that allows T. pallidum to establish lifelong infection. 55 56 57 58 Syphilis, caused by the spirochete Treponema pallidum subspecies pallidum (T. 59 pallidum), is a significant global health problem. Although most syphilis cases occur in 60 low-income countries, where the disease is endemic, rates of syphilis infection have 61 been steadily increasing for the last two decades in high-income nations, particularly in 62 men who have sex with men (MSM) and HIV-infected individuals [1-4]. Syphilis is an 63 acute and chronic sexually transmitted infection marked by distinct early and late stages 64 [5]. These stages are generally distinguished by unique clinical manifestations with 65 symptoms associated with the late stage developing up to several decades after initial 66 infection and following a long period of disease latency [6]. 67 The mechanisms that allow T. pall...