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

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 x 10(4) 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 10(8) 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.     

Borrelia burgdorferi complement regulator-acquiring surface proteins (BbCRASPs): Expression patterns during the mammal-tick infection cycle

Host complement is widely distributed throughout mammalian body fluids and can be activated immediately as part of the first line of defense against invading pathogens. The agent of Lyme disease, Borrelia burgdorferi sensu lato (s.l.), is naturally resistant to that innate immune defense system of its hosts. One resistance mechanism appears to involve binding fluid-phase regulators of complement to distinct borrelial outer surface molecules known as CRASPs (complement regulator acquiring surface proteins). Using sensitive molecular biology techniques, expression patterns of all three classes of genes encoding the CRASPs of B. burgdorferi sensu stricto (BbCRASPs) have been analyzed throughout the natural tick-mammal infection cycle. Each class shows a different expression profile in vivo and the results are summarized herein. Studies on the expression of B. burgdorferi genes using animal models of infection have advanced our knowledge on the ability of the causative agent to circumvent innate immune defenses, the contributions of CRASPs to spirochete infectivity, and the pathogenesis of Lyme disease.     

Borrelia burgdorferi binding of host complement regulator factor H is not required for efficient mammalian infection

The causative agent of Lyme disease, Borrelia burgdorferi, is naturally resistant to its host's alternative pathway of complement-mediated killing. Several different borrelial outer surface proteins have been identified as being able to bind host factor H, a regulator of the alternative pathway, leading to a hypothesis that such binding is important for borrelial resistance to complement. To test this hypothesis, the development of B. burgdorferi infection was compared between factor H-deficient and wild-type mice. Factor B- and C3-deficient mice were also studied to determine the relative roles of the alternative and classical/lectin pathways in B. burgdorferi survival during mammalian infection. While it was predicted that B. burgdorferi should be impaired in its ability to infect factor H-deficient animals, quantitative analyses of bacterial loads indicated that those mice were infected at levels similar to those of wild-type and factor B- and C3-deficient mice. Ticks fed on infected factor H-deficient or wild-type mice all acquired similar numbers of bacteria. Indirect immunofluorescence analysis of B. burgdorferi acquired by feeding ticks from the blood of infected mice indicated that none of the bacteria had detectable levels of factor H on their outer surfaces, even though such bacteria express high levels of surface proteins capable of binding factor H. These findings demonstrate that the acquisition of host factor H is not essential for mammalian infection by B. burgdorferi and indicate that additional mechanisms are employed by the Lyme disease spirochete to evade complement-mediated killing.     

Protection elicited by native outer membrane protein Oms66 (p66) against host-adapted Borrelia burgdorferi: conformational nature of bactericidal epitopes

Oms66 is a Borrelia burgdorferi outer membrane porin protein whose role in Lyme disease pathogenesis and immunity has not been well established. Oms66 was solubilized from whole-cell lysates of strain B313 (which is derived from B31 but lacks OspA, -B, -C, and -D) and purified to homogeneity by fast-protein liquid chromatography. Purified native Oms66 (nOms66), which retained the ability to form large channels in a planar lipid bilayer model membrane system, and denatured Oms66 (hOms66) were used to immunize New Zealand White rabbits. The resulting Oms66 antisera were tested in a complement-dependent borreliacidal assay in parallel with basal serum and with serum from rabbits immune to reinfection with B. burgdorferi (IRS). IRS showed high-titer complement-dependent killing of both strains B31 and B313. Sera from animals immunized with nOms66 showed high-titer complement-dependent killing activity against strain B313 but exhibited no killing of B31. By comparison, serum generated from immunizations with hOms66 showed no killing activity against either strain. Following adsorption of antiserum to nOms66 with recombinant Oms66 (rOms66), the serum antibodies no longer bound to rOms66 or to nOms66 that had been denatured with 8 M urea. However, the antibodies still bound to nOms66 and killing activity against B313 was retained, thus suggesting that native, conformational epitopes are targets of this bactericidal activity. Six C3H HeJ mice were immunized with nOms66 and were challenged using "host-adapted" B. burgdorferi B31 by skin implantation of infected mouse ear tissue. Four of the six mice were protected against both localized and disseminated infection. These findings indicate that native Oms66 can elicit potent bactericidal activity and significant protective immunity against host-adapted organisms.     

Interaction of Lyme disease spirochetes with cultured eucaryotic cells.

The association of the Lyme disease spirochete, Borrelia burgdorferi, with cultured human endothelial cells was investigated. Attachment was time and temperature dependent, with optimal adherence occurring after 4 h of incubation at 37 degrees C. Pretreatment of borreliae with heat, immune human serum, or monoclonal antibodies directed against outer surface protein B (OspB) reduced the attachment of organisms to host cell monolayers. These results suggest that the adherence of B. burgdorferi may be mediated, at least in part, by borrelial surface proteins.     

Autoimmunity in Lyme disease: molecular cloning of antigens recognized by antibodies in the cerebrospinal fluid

In inflammatory disease of the central nervous system (CNS), oligoclonal bands of immunoglobulin with restricted heterogeneity can often be observed in cerebrospinal fluid (CSF) samples. These antibodies can be directed against the disease inducing pathogen or might be autoreactive and involved in the process of brain inflammation and demyelination. We used a molecular biology approach to characterize these antibody responses in patients with Lyme disease. This disorder is caused by infections with the spirochete Borrelia burgdorferi which is transmitted by ticks. Lyme disease can be associated with neurological symptoms due to inflammation of the central and peripheral nervous system. Phage lambda gtll expression libraries from B. burgdorferi and human brain were screened with cerebrospinal fluid antibody probes from patients with Lyme disease. We obtained recombinant phage clones encoding antigenic proteins from both B. burgdorferi and human CNS libraries. Thus, in this study two patients with chronic Lyme disease produced antibodies against recombinant B. burgdorferi as well as against CNS proteins, and the generation of this transient autoimmune response might be essential to the development of demyelinating disease.     

A chromosomal Borrelia burgdorferi gene encodes a 22-kilodalton lipoprotein, P22, that is serologically recognized in Lyme disease.

We describe the isolation of the gene encoding a 22-kDa antigen from Borrelia burgdorferi, the etiologic agent of Lyme disease. The p22 gene is 582 nucleotides in length and encodes a protein of 194 amino acids with a predicted molecular mass of 21.8 kDa. The leader signal sequence of P22 consists of a positively charged short amino terminus, a central hydrophobic domain, and at the carboxyl terminus, a cleavage site that is presumably recognized and cleaved by a B. burgdorferi signal peptidase. P22 has 98.5% homology with the recently described B. burgdorferi protein IpLA7. P22 is processed as a lipoprotein, as demonstrated by [3H]palmitate labeling. Pulsed-field gel electrophoresis showed that p22, like LA7, is localized to the linear chromosome of B. burgdorferi. Examination of sera from patients with Lyme disease revealed that antibodies to P22 are rarely detected in patients with early-stage disease characterized by erythema migrans (2 of 20), and 35% of the patients with late-stage disease characterized by arthritis (9 of 26) developed antibodies to P22. Sera from patients with syphilis did not react with P22. When patients with late-stage disease were tested for their antibody reactivities to four other outer surface proteins (OspA), OspB, OspE, and OspF), 75% of these patients responded to P22 or to one or more outer surface proteins.     

Variable serum immunoglobulin responses against different Borrelia burgdorferi sensu lato species in a population at risk for and patients with Lyme disease.

Borrelia burgdorferi sensu lato species display considerable antigenic polymorphism. In order to evaluate the importance of this antigenic heterogeneity in the serodiagnosis of Lyme disease, the serum immunoglobulin G response in 148 healthy individuals from an area in northern Sweden where Lyme disease is endemic and in 40 American patients with Lyme disease was assessed. In a seroprevalence study, the control group included 173 individuals from a region of northern Sweden where Lyme disease is not endemic. The two enzyme immunoassays used were based on outer membrane-associated proteins of either B. burgdorferi sensu stricto or Borrelia garinii. The Swedish populations were also screened for antiflagellum seroreactivity. The individuals from the area of endemicity were significantly more seropositive for the subcellular protein fraction of the local B. garinii isolate NBS16 than the control group (11.5 versus 2.9%; P = 0.005) but were not significantly more positive for the other antigens used. In contrast, American patients with Lyme disease were significantly more reactive against the North American B. burgdorferi sensu stricto strain B31 than against B. garinii NBS16 (57.5 versus 15.0%; P = 0.0001). Immunoblot analysis suggests that the borrelial outer surface protein C is involved in triggering the production of species-specific antibody during localized Lyme disease. We conclude that a species-specific immune response develops during infection with Lyme disease Borrelia spp. Thus, the reliability of a serological investigation of Lyme disease increases when one measures antibody titers against the outer membrane proteins of Lyme disease Borrelia spp. occurring in a particular geographic region.     

Cloning and Molecular Characterization of Plasmid-Encoded Antigens of Borrelia burgdorferi

Thirteen independent clones that encode Borrelia burgdorferi antigens utilizing antiserum from infection-immune rabbits were identified. The serum was adsorbed against noninfectious B. burgdorferi B31 to enrich for antibodies directed against either infection-associated antigens of B. burgdorferi B31 or proteins preferentially expressed during mammalian infection. The adsorption efficiency of the immune rabbit serum (IRS) was assessed by Western immunoblot analysis with protein lysates derived from infectious and noninfectious B. burgdorferi B31. The adsorbed IRS was used to screen a B. burgdorferi expression library to identify immunoreactive phage clones. Clones were then expressed in Escherichia coli and subsequently analyzed by Western blotting to determine the molecular mass of the recombinant B. burgdorferi antigens. Southern blot analysis of the 13 clones indicated that 10 contained sequences unique to infectious B. burgdorferi. Nucleotide sequence analysis indicated that the 13 clones were composed of 9 distinct genetic loci and that all of the genes identified were plasmid encoded. Five of the clones carried B. burgdorferi genes previously identified, including those encoding decorin binding proteins A and B (dbpAB), a rev homologue present on the 9-kb circular plasmid (cp9), a rev homologue from the 32-kb circular plasmid (cp32-6), erpM, and erpX. Additionally, four previously uncharacterized loci with no known homologues were identified. One of these unique clones encoded a 451-amino-acid lipoprotein with 21 consecutive, invariant 9-amino-acid repeats near the amino terminus that we have designated VraA (for "virulent strain-associated repetitive antigen A"). Since all the antigens identified are recognized by serum from infection immune rabbits, these antigens represent potential vaccine candidates and, based on the identification of dbpAB in this screen, may also be involved in pathogenic processes operative in Lyme borreliosis.     

Partial destruction of Borrelia burgdorferi within ticks that engorged on OspE- or OspF-immunized mice.

We determined whether Borrelia burgdorferi outer surface proteins (Osps) E and F could elicit immune responses useful for a Lyme disease vaccine. Thirty days after challenge with B. burgdorferi, mice produced antibodies to OspE but not OspF, whereas antibodies to OspF were present in sera of mice obtained 90 days after infection. Examination of sera from patients with Lyme disease revealed antibodies to OspF in a small number (14%) of early-stage disease patients but in a majority (58%) of patients with late-stage disease, while antibodies to OspE were rarely detected in patients. Mice immunized with recombinant OspE or OspF produced high titers of antibodies to OspE or OspF, respectively. OspF-immunized mice were partially protected from both intradermal syringe challenge and tick-mediated transmission of B. burgdorferi while vaccination with OspE did not confer immunity. B. burgdorferi organisms were, however, substantially destroyed within ticks that engorged on either OspE- (75% reduction in the number of spirochetes within the ticks, compared with controls) or OspF (90% reduction in the number of spirochetes within the ticks)-immunized mice.     

Abilities of OspA proteins from different seroprotective groups of Borrelia burgdorferi to protect hamsters from infection.

The ability of vaccination with recombinant OspA from six seroprotective groups of Borrelia burgdorferi sensu lato to induce protection against infection with homologous and other Lyme spirochetes was examined in hamsters. Antisera generated against the OspA proteins of B. burgdorferi sensu stricto S-1-10 and C-1-11 (seroprotective groups 1 and 2, respectively), Borrelia afzelii BV1 (seroprotective group 4), and Borrelia garinii LV4 (seroprotective group 5) were able to kill the homologous spirochete in vitro but not other isolates. Surprisingly, antisera against B. afzelii PKo (seroprotective group 6) and B. burgdorferi sensu lato LV5 (seroprotective group 3) OspA proteins were unable to kill the homologous organism, although LV5 OspA antisera killed the heterologous isolates S-1-10 and LV4. In vivo vaccination studies supported the in vitro findings, confirming that vaccination with a single OspA protein does not provide complete protection against challenge with all Lyme disease spirochetes. In addition, OspA antibodies from some isolates may not protect against the homologous isolate. The induction of protective antibodies against other B. burgdorferi proteins may be necessary to insure a comprehensive Lyme disease vaccine.     

Expression of complement factor H binding immunoevasion proteins in Borrelia garinii isolated from patients with neuroborreliosis

The Lyme disease-pathogen Borrelia burgdorferi binds the complement inhibitor factor H (FH) to its outer surface protein E- (OspE) and BbA68-families of lipoproteins. In earlier studies, only serum-resistant strains of the genospecies B. burgdorferi sensu stricto or B. afzelii, but not serum-sensitive B. garinii strains, have been shown to bind FH. Since B. garinii often causes neuroborreliosis in man, we have readdressed the interactions of B. garinii with FH. B. garinii 50/97 strain did not express FH-binding proteins. By transforming the B. garinii 50/97 strain with an OspE-encoding gene from complement-resistant B. burgdorferi (ospE-297), its resistance to serum killing could be increased. OspE genes were detected and cloned from the B. garinii BITS, Pistoia and 40/97 strains by PCR and sequencing. The deduced amino acid sequences differed in an N-terminal lysine-rich FH-binding region from OspE sequences of resistant strains. Recombinant B. garinii BITS OspE protein was found to have a considerably lower FH-binding activity than the B. burgdorferi sensu stricto 297 OspE protein P21 (P21-297). Unlike bacteria that had been kept in culture for a long time, neurovirulent B. garinii strains from neuroborreliosis patients were found to express approximately 27-kDa FH-binding proteins. These were not recognized by polyclonal anti-OspE or anti-BbA68 antibodies. We conclude that B. garinii strains carry ospE genes but have a decreased expression of OspE proteins and a reduced ability to bind FH, especially when grown for prolonged periods in vitro. Recently isolated neuroinvasive B. garinii strains, however, can express FH-binding proteins, which may contribute to the virulence of neuroborreliosis-causing B. garinii strains.     

Changes in temporal and spatial patterns of outer surface lipoprotein expression generate population heterogeneity and antigenic diversity in the Lyme disease spirochete,Borrelia burgdorferi

Borrelia burgdorferi differentially expresses many of the OspE/F/Elp paralogs during tick feeding. These findings, combined with the recent report that stable B. burgdorferi infection of mammals occurs only after 53 h of tick attachment, prompted us to further analyze the expression of the OspE/F/Elp paralogs during this critical period of transmission. Indirect immunofluorescence analysis revealed that OspE, p21, ElpB1, ElpB2, and OspF/BbK2.11 are expressed in the salivary glands of ticks allowed to feed on mice for 53 to 58 h. Interestingly, many of the spirochetes in the salivary glands that expressed abundant amounts of these antigens were negative for OspA and OspC. Although prior reports have indicated that OspE/F/Elp orthologs are surface exposed, none of the individual lipoproteins or combinations of the lipoproteins protected mice from challenge infections. To examine why these apparently surface-exposed lipoproteins were not protective, we analyzed their genetic stability during infection and their cellular locations after cultivation in vitro and within dialysis membrane chambers, mimicking a mammalian host-adapted state. Combined restriction fragment length polymorphism and nucleotide sequence analyses revealed that the genes encoding these lipoproteins are stable for at least 8 months postinfection. Interestingly, cellular localization experiments revealed that while all of these proteins can be surface localized, there were significant populations of spirochetes that expressed these lipoproteins only in the periplasm. Furthermore, host-specific signals were found to alter the expression patterns and final cellular location of these lipoproteins. The combined data revealed a remarkable heterogeneity in populations of B. burgdorferi during tick transmission and mammalian infection. The diversity is generated not only by temporal changes in antigen expression but also by modulation of the surface lipoproteins during infection. The ability to regulate the temporal and spatial expression patterns of lipoproteins throughout infection likely contributes to persistent infection of mammals by B. burgdorferi.     

The thermophilic, homohexameric aminopeptidase of Borrelia burgdorferi is a member of the M29 family of metallopeptidases

Proteases are implicated in several aspects of the physiology of microorganisms, as well as in host-pathogen interactions. Aminopeptidases are also emerging as novel drug targets in infectious agents. In this study, we have characterized an aminopeptidase from the spirochete Borrelia burgdorferi, the causative agent of Lyme disease. The aminopeptidolytic activity was identified in cell extracts from B. burgdorferi by using the substrate leucine-7-amido-4-methylcoumarin. A protein displaying this activity was purified from B. burgdorferi by a two-step chromatographic procedure, yielding a approximately 300-kDa homo-oligomeric enzyme formed by monomers of approximately 50 kDa. Gel enzymography experiments showed that enzymatic activity depends on the oligomeric structure of the protease but does not involve interchain disulfide bonds. The enzyme was identified by peptide mass fingerprinting as the putative aminopeptidase II of B. burgdorferi, encoded by the gene BB0069. It shares significant identity to members of the M29/T family of metallopeptidase, is sensitive to bestatin, has a neutral pH optimum, and displays maximal activity at 60 degrees C. Its activity is 1.75-fold higher at the temperature of the mammalian host than at that of the insect host of the pathogen. The activity of this thermophilic aminopeptidase of B. burgdorferi (TAP(Bb)) depends on Zn2+, and temperatures over 70 degrees C promoted its inactivation through a transition from the hexameric state to the monomeric state. Since B. burgdorferi is deficient in pathways for amino acid synthesis, TAP(Bb) could play a role in supplying required amino acids. Alternatively, the enzyme could be involved in peptide and/or protein processing.