Ellen S. Shang

Virulent strain associated outer membrane proteins of Borrelia burgdorferi.

We have isolated and purified outer membrane vesicles (OMV) from Borrelia burgdorferi strain B31 based on methods developed for isolation of Treponema pallidum OMV. Purified OMV exhibited distinct porin activities with conductances of 0.6 and 12.6 nano-Siemen and had no detectable beta-NADH oxidase activity indicating their outer membrane origin and their lack of inner membrane contamination, respectively. Hydrophobic proteins were identified by phase partitioning with Triton X-114. Most of these hydrophobic membrane proteins were not acylated, suggesting that they are outer membrane-spanning proteins. Identification of palmitate-labeled lipoproteins revealed that several were enriched in the OMV, several were enriched in the protoplasmic cylinder inner membrane fraction, and others were found exclusively associated with the inner membrane. The protein composition of OMV changed significantly with successive in vitro cultivation of strain B31. Using antiserum with specificity for virulent strain B31, we identified OMV antigens on the surface of the spirochete and identified proteins whose presence in OMV could be correlated with virulence and protective immunity in the rabbit Lyme disease model. These virulent strain associated outer membrane-spanning proteins may provide new insight into the pathogenesis of Lyme disease.

Comparison of Protection in Rabbits against Host-Adapted and Cultivated Borrelia burgdorferi following Infection-Derived Immunity or Immunization with Outer Membrane Vesicles or Outer Surface Protein A

ABSTRACT In this study, infection-derived immunity in the rabbit model of Lyme disease was compared to immunity following immunization with purified outer membrane vesicles (OMV) isolated from Borrelia burgdorferi and recombinant outer surface protein A (OspA). Immunization of rabbits with OMV isolated from virulent strain B31 and its avirulent derivative B313 (lacking OspA and DbpA) conferred highly significant protection against intradermal injection with 6 × 104 in vitro-cultivated virulent B. burgdorferi. This is the first demonstration of protective immunogenicity induced by OMV. While immunization with OspA and avirulent B31 OMV provided far less protection against this challenge, rabbits with infection-derived immunity were completely protected. Protection against host-adapted B. burgdorferi was assessed by implantation of skin biopsies taken from rabbit erythema migrans (a uniquely rich source of B. burgdorferi in vertebrate tissue) containing up to 108 spirochetes. While all of the OMV- and OspA-immunized rabbits were fully susceptible to skin and disseminated infection, rabbits with infection-derived immunity were completely protected. Analysis of the antibody responses to outer membrane proteins, including DbpA, OspA, and OspC, suggests that the remarkable protection exhibited by the infection-immune rabbits is due to antibodies directed at antigens unique to or markedly up-regulated in host-adapted B. burgdorferi.

Immunohistochemical Analysis of Lyme Disease in the Skin of Naive and Infection-Immune Rabbits following Challenge

ABSTRACT In this study, skin histopathology from naive and infection-derived immune rabbits was compared following intradermal challenge usingBorrelia burgdorferi B31 strain. The presence or absence of spirochetes in relationship to host cellular immune responses was determined from the time of intradermal inoculation to the time of erythema migrans (EM) development (∼7 days in naive rabbits) and through development of challenge immunity (∼5 months in naive rabbits). Skin biopsies were obtained and analyzed for the presence of spirochetes, B cells, T cells, polymorphonuclear cells (PMNs), and macrophages by immunohistochemical techniques. In infected naive animals, morphologically identifiable spirochetes were detected at 2 h and up to 3 weeks postinfection. At 12 and 24 h postinfection there was a marked PMN response that decreased by 36 to 48 h; by 72 h the PMNs were replaced by a few infiltrating macrophages. At the time of EM development and 14 days postinfection, the PMNs and macrophages were replaced by a lymphocytic infiltrate. There was a greater number of spirochetes at 14 days, a time when EM had resolved, than at 7 days postinfection. By 3 weeks postinfection there were few organisms and lymphocytes detectable. In contrast to infected naive rabbits, intact spirochetes were never visualized in skin biopsies from infection-immune rabbits; only spirochetal antigen was detected at 2, 12, and 24 h in the presence of a numerous PMN infiltrate. By 36 h postchallenge, spirochetal antigen could not be detected and the PMN response was replaced by a few infiltrating macrophages. By 72 h postchallenge, PMNs and macrophages were absent from the skin; B and T cells were never detected at any time point in skin from infection-immune rabbits. The destruction of spirochetes in immune animals in the presence of PMNs and in the absence of a lymphocytic infiltrate suggests that infection-derived immunity is antibody mediated.

Homologous and Heterologous Borrelia burgdorferi Challenge of Infection-Derived Immune Rabbits Using Host-Adapted Organisms

ABSTRACT We have recently found that strain B31 infection-immune rabbits are completely protected against homologous challenge with large numbers (>106) of host-adapted Borrelia burgdorferi(HAB) (E. S. Shang, C. I. Champion, X. Wu, J. T. Skare, D. B. Blanco, J. N. Miller, and M. A. Lovett, Infect. Immun. 68:4189–4199, 2000). In this study, we have extended these findings to determine whether B31 strain infection-immune rabbits are also protected against heterologous HAB challenge. Infection-immune rabbits challenged with large numbers (>106) of homologous HAB strain B31 were completely protected from erythema migrans (EM) and skin and disseminated infection. In contrast, infection-immune rabbits challenged with heterologous HAB strains N40 and Sh-2-82 were completely susceptible to EM and skin and disseminated infection; challenge with strain 297 also resulted in EM and infection of the skin and viscera, but clearance of infection occurred 3 weeks postchallenge. These findings confirm that immunity elicited in rabbits by B31 strain infection confers complete protection against large-dose homologous HAB challenge but not against a heterologous strain.