Active and Passive Immunity against Borrelia burgdorferi Decorin Binding Protein A (DbpA) Protects against Infection

Borrelia burgdorferi, the spirochete that causes Lyme disease, binds decorin, a collagen-associated extracellular matrix proteoglycan found in the skin (the site of entry for the spirochete) and in many other tissues. Two borrelial adhesins that recognize this proteoglycan, decorin binding proteins A and B (DbpA and DbpB, respectively), have recently been identified. Infection of mice by low-dose B. burgdorferi challenge elicited antibodies against DbpA and DbpB that were sustained at high levels, suggesting that these antigens are expressed in vivo. Scanning immunoelectron microscopy showed that DbpA was surface accessible on intact borreliae. Passive administration of DbpA antiserum protected mice from infection following challenge with heterologous B. burgdorferi sensu stricto isolates, even when serum administration was delayed for up to 4 days after challenge. DbpA is the first antigen target identified that is capable of mediating immune resolution of early, localized B. burgdorferi infections. DbpA immunization also protected mice from B. burgdorferi challenge; DbpB immunization was much less effective. DbpA antiserum inhibited in vitro growth of many B. burgdorferi sensu lato isolates of diverse geographic, phylogenetic, and clinical origins. In combination, these findings support a role for DbpA in the immunoprophylaxis of Lyme disease and suggest that DbpA vaccines have the potential to eliminate early-stage B. burgdorferi infections.     

Humoral Immunity to Borrelia burgdorferi N40 Decorin Binding Proteins during Infection of Laboratory Mice

A Borrelia burgdorferi N40 genomic expression library was screened with serum from actively infected mice to identify gene products that elicit protective immunity. A clone that contained a putative bicistronic operon containing two genes that encoded 20- and 22-kDa lipoproteins was identified and sequenced. These genes showed homology with the genes encoding decorin binding proteins DbpB and DbpA, respectively, of B. burgdorferi 297 and B31. N40-dbpA DNA hybridized with B. burgdorferi N40 DNA on a single 48-kb linear plasmid. Homologous genes could be amplified under various degrees of stringency by PCR or hybridized by Southern blotting from B. burgdorferi sensu stricto N40 and B31, and from B. burgdorferi sensu lato PBi and 25015, but not PKo. Recombinant N40-DbpB and N40-DbpA were reactive with antibody in serum from infected mice, and serum was more reactive against N40-DbpA than against B. burgdorferi N40 recombinant P39, OspC, or OspA. Sera from mice infected with B. burgdorferi sensu lato strains PKo and PBi were weakly reactive against N40-DbpB and N40-DbpA, and sera from mice infected with 25015 were moderately reactive, compared to sera from mice infected with B. burgdorferi N40. Hyperimmunization of mice with N40-DbpA, but not N40-DbpB, induced protective immunity against syringe challenge with cultured B. burgdorferi N40. DbpA may therefore be one of the antigens responsible for eliciting protective antibody known to exist in serum from infected mice. DNA amplification and serology suggest that DbpB and DbpA are likely to have homologs throughout the B. burgdorferi sensu lato family, but they are likely to be heterogeneous.     

Gamma Interferon Inhibits Production of Anti-OspA Borreliacidal Antibody In Vitro

The ability of a Lyme borreliosis vaccine to induce and maintain sustained levels of borreliacidal antibody is necessary for prolonged protection against infection with Borrelia burgdorferi. Vaccination against infection with B. burgdorferi could be improved by determining the mechanism(s) that influences the production of protective borreliacidal antibody. Borreliacidal antibody was inhibited in cultures of lymph node cells obtained from C3H/HeJ mice vaccinated with formalin-inactivated B. burgdorferi and cultured with macrophages and B. burgdorferi and treated with recombinant gamma interferon (rIFN-γ). The suppression of production of outer surface protein A (OspA) borreliacidal antibody by rIFN-γ was not affected by the time of treatment. In addition, treatment with rIFN-γ inhibited the production of other anti-B. burgdorferi antibodies. By contrast, treatment of cultures of immune lymph node cells with anti-IFN-γ marginally increased the production of borreliacidal antibody and enhanced the production of other antibodies directed against B. burgdorferi. These results show that IFN-γ does not play a major role in the production of anti-OspA borreliacidal antibody. Additional studies are needed to determine which cytokine(s) will enhance production of borreliacidal antibody.     

Both decorin-binding proteins A and B are critical for the overall virulence of Borrelia burgdorferi

Both decorin-binding proteins (DbpA and DbpB) of the Lyme disease spirochete Borrelia burgdorferi bind decorin and glycosaminoglycans, two important building blocks of proteoglycans that are abundantly found in the extracellular matrix (ECM) and connective tissues as well as on cell surfaces of mammals. As an extracellular pathogen, B. burgdorferi resides primarily in the ECM and connective tissues and between host cells during mammalian infection. The interactions of B. burgdorferi with these host ligands mediated by DbpA and DbpB potentially influence various aspects of infection. Here, we show that both DbpA and DbpB are critical for the overall virulence of B. burgdorferi in the murine host. Disruption of the dbpBA locus led to nearly a 10⁴-fold increase in the 50% infectious dose (ID₅₀). Complementation of the mutant with either dbpA or dbpB reduced the ID₅₀ from over 10⁴ to roughly 10³ organisms. Deletion of the dbpBA locus affected colonization in all tissues of infected mice. The lack of dbpA alone precluded the pathogen from colonizing the heart tissue, and B. burgdorferi deficient for DbpB was recovered only from 42% of the heart specimens of infected mice. Although B. burgdorferi lacking either dbpA or dbpB was consistently grown from joint specimens of almost all infected mice, it generated bacterial loads significantly lower than the control. The deficiency in either DbpA or DbpB did not reduce the bacterial load in skin, but lack of both significantly did. Taken together, the study results indicate that neither DbpA nor DbpB is essential for mammalian infection but that both are critical for the overall virulence of B. burgdorferi.     

Molecular Analysis of Sequence Heterogeneity among Genes Encoding Decorin Binding Proteins A and B of Borrelia burgdorferi Sensu Lato

Immunization of mice with Borrelia burgdorferi decorin binding protein A (DbpA), one of two gene products of the dbpBA locus, has been shown recently to confer protection against challenge. Hyperimmune DbpA antiserum killed a large number of B. burgdorferi sensu lato isolates of diverse phylogeny and origin, suggesting conservation of the protective epitope(s). In order to evaluate the heterogeneity of DbpA and DbpB and to facilitate defining the conserved epitope(s) of these antigens, the sequences of the dbpA genes from 29 B. burgdorferi sensu lato isolates and of the dbpB genes from 15 B. burgdorferi sensu lato isolates were determined. The predicted DbpA sequences were fairly heterogeneous among the isolates (58.3 to 100% similarity), but DbpA sequences with the highest similarity tended to group into species previously defined by well-characterized chromosomal markers. In contrast, the predicted DbpB sequences were highly conserved (96.3 to 100% similarity). Substantial diversity in DbpA sequence was seen among isolates previously shown to be killed by antiserum against a single DbpA, suggesting that one or more conserved protective epitopes are composed of noncontiguous amino acids. The observation of individual dbpA alleles with sequence elements characteristic of more than one B. burgdorferi sensu lato species was consistent with a role for genetic recombination in the generation of dbpA diversity.     

Borreliacidal OspC Antibodies Specific for a Highly Conserved Epitope Are Immunodominant in Human Lyme Disease and Do Not Occur in Mice or Hamsters

Humans produce highly specific borreliacidal antibodies against outer surface protein C (OspC) shortly after infection with Borrelia burgdorferi sensu stricto. We previously demonstrated the epitope recognized by immunoglobulin M (IgM) and IgG OspC borreliacidal antibodies was located within the 50 amino acids nearest the carboxy (C) terminus. In this study, we show the immunodominant epitope is located in the highly conserved region within the seven C-terminal amino acids. Six early Lyme disease sera that contained borreliacidal activity and IgM and/or IgG OspC antibodies were chosen randomly and adsorbed with truncated OspC containing the 16 or 7 amino acids nearest the C terminus. Adsorptions with each truncated protein abrogated the borreliacidal activity completely. In addition, only small concentrations of OspC antibodies remained detectable by enzyme-linked immunosorbent assay and Western blotting. Moreover, borreliacidal OspC antibodies were not induced in laboratory mice or hamsters despite heavy infections with B. burgdorferi spirochetes. These findings confirm that borreliacidal antibodies comprise the majority of the IgM and IgG OspC antibody response in human Lyme disease and that the epitope is located in the highly conserved C terminus. In addition, rodent animal models appear to be inappropriate subjects for assessing the effectiveness of the epitope for serodiagnosis or as a human Lyme disease vaccine.     

Evaluation of the Borrelia burgdorferi BBA64 Protein as a Protective Immunogen in Mice

The Borrelia burgdorferi bba64 gene product is a surface-localized lipoprotein synthesized within mammalian and tick hosts and is involved in vector transmission of disease. These properties suggest that BBA64 may be a vaccine candidate against Lyme borreliosis. In this study, protective immunity against B. burgdorferi challenge was assessed in mice immunized with the BBA64 protein. Mice developed a high-titer antibody response following immunization with soluble recombinant BBA64 but were not protected when challenged by needle inoculation of culture-grown spirochetes. Likewise, mice passively immunized with an anti-BBA64 monoclonal antibody were not protected against needle-inoculated organisms. BBA64-immunized mice were subjected to B. burgdorferi challenge by the natural route of a tick bite, but these trials did not demonstrate significant protective immunity in either outbred or inbred strains of mice. Lipidated recombinant BBA64 produced in Escherichia coli was assessed for possible improved elicitation of a protective immune response. Although inoculation with this antigen produced a high-titer antibody response, the lipidated BBA64 also was unsuccessful in protecting mice from B. burgdorferi challenge by tick bites. Anti-BBA64 antibodies raised in rats eradicated the organisms, as evidenced by in vitro borreliacidal assays, thus demonstrating the potential for BBA64 to be effective as a protective immunogen. However, passive immunization with the same monospecific rat anti-BBA64 polyclonal serum failed to provide protection against tick bite-administered challenge. These results reveal the challenges faced in not only identifying B. burgdorferi proteins with potential protective capability but also in producing recombinant antigens conducive to preventive therapies against Lyme borreliosis.     

Influence of Outer Surface Protein A Antibody on Borrelia burgdorferi within Feeding Ticks

Borrelia burgdorferi, the spirochetal agent of Lyme disease, is transmitted by Ixodes ticks. When an infected nymphal tick feeds on a host, the bacteria increase in number within the tick, after which they invade the tick’s salivary glands and infect the host. Antibodies directed against outer surface protein A (OspA) of B. burgdorferi kill spirochetes within feeding ticks and block transmission to the host. In the studies presented here, passive antibody transfer experiments were carried out to determine the OspA antibody titer required to block transmission to the rodent host. OspA antibody levels were determined by using a competitive enzyme-linked immunosorbent assay that measured antibody binding to a protective epitope defined by monoclonal antibody C3.78. The C3.78 OspA antibody titer (>213 μg/ml) required to eradicate spirochetes from feeding ticks was considerably higher than the titer (>6 μg/ml) required to block transmission to the host. Although spirochetes were not eradicated from ticks at lower antibody levels, the antibodies reduced the number of spirochetes within the feeding ticks and interfered with the ability of spirochetes to induce ospC and invade the salivary glands of the vector. OspA antibodies may directly interfere with the ability of B. burgdorferi to invade the salivary glands of the vector; alternately, OspA antibodies may lower the density of spirochetes within feeding ticks below a critical threshold required for initiating events linked to transmission.     

Inhibition of the Production of Anti-OspA Borreliacidal Antibody with T Cells from Hamsters Vaccinated against Borrelia burgdorferi

The serious morbidity associated with Lyme borreliosis has focused considerable effort on the development of a comprehensive vaccine for protection against infection with Borrelia burgdorferi. Induction of borreliacidal antibody by vaccination or infection has been shown to correlate with protection of humans and animals against infection with the Lyme spirochete. In this report, we showed that high levels of borreliacidal antibody (titer of 1,280) were produced in vitro when T and B cells from hamsters 14 days after vaccination were incubated with macrophages and B. burgdorferi. By contrast, T and B cells from hamsters 7 or 21 days after vaccination failed to initiate production of borreliacidal activity. Furthermore, the T cells from hamsters 7 or 21 days after vaccination inhibited the in vitro production of borreliacidal antibody when cocultured with T and B cells obtained from hamsters 14 days after vaccination. When cell-free supernatants from the suspensions of T and B cells from hamsters 14 days after vaccination were absorbed with recombinant OspA, they lost nearly all borreliacidal activity. The removal of anti-OspA antibody resulted in a decrease in borreliacidal titer from 1,280 to less than 4. These results demonstrate that T cells from vaccinated animals can prevent a sustained production of protective borreliacidal antibody.     

Neutralization of Gamma Interferon Augments Borreliacidal Antibody Production and Severe Destructive Lyme Arthritis in C3H/HeJ Mice

Development of a high level of sustained borreliacidal antibody is paramount for maintaining protection against infection with Borrelia burgdorferi. We show that production of borreliacidal antibody can be enhanced by preventing the effects of gamma interferon (IFN-γ). When lymph node cells capable of producing borreliacidal antibody were cultured with anti-murine IFN-γ, an eightfold increase in borreliacidal antibody production was obtained. However, anti-IFN-γ treatment of these cells also enhanced their ability to adaptively induce arthritis. When anti-IFN-γ-treated lymph node cells producing borreliacidal antibody were infused into C3H/HeJ mice and the mice were then challenged with B. burgdorferi, the mice developed severe destructive Lyme arthritis. Additional studies are needed to delineate the immune response responsible for the induction of arthritis and production of borreliacidal antibody. These studies are needed to ensure an effective and safe vaccine against infection with B. burgdorferi.     

Decorin Binding Proteins A and B in the Serodiagnosis of Lyme Disease in North America

The laboratory diagnosis of Lyme disease is based upon the detection of antibodies generated against Borrelia burgdorferi using a two-tier assay, typically consisting of an enzyme-linked immunosorbent assay (ELISA), followed by a Western blot. This system, put into place to address the nonspecificity associated with standalone first-tier assays, is insensitive for diagnosing early infection, when most people seek care. The use of bacterial lysates or whole-protein antigens as first-tier assay targets contributes to nonspecificity due, in part, to the presence of cross-reactive epitopes that are also found in other bacteria. This precludes their use as sensitive standalone assays. The use of peptides containing linear epitopes that are highly specific for B. burgdorferi offers a method for reducing this cross-reactivity. In the present study, we mapped the linear epitopes of the prominently expressed Borrelia adhesins decorin binding protein A (DbpA) and DbpB. We identified several epitopes in each protein that were highly conserved among North American strains of B. burgdorferi, and we screened peptides containing specific epitopes using serum panels from early and late Lyme disease patients. The individual peptides primarily detected IgM but not IgG, while the proteins efficiently detected both IgM and IgG. While no individual peptide demonstrated better utility for antibody detection than its respective whole protein, an assay containing a combination of a DbpA and a DbpB peptide adequately detected both IgM and IgG, accurately identifying 87.5% (84/96) of the early Lyme disease patients and 80.0% (16/20) of the late Lyme disease patients.     

Circumvention of outer surface protein A immunity by host-adapted Borrelia burgdorferi.

Outer surface protein A (OspA), which is abundantly expressed in cultured Borrelia burgdorferi, appears to be down-regulated or masked following low-dose infection, and OspA immunization did not prevent infection, dissemination, or disease development with host-adapted spirochetes. Seroconversion of mice to B. burgdorferi OspA depended on dose and viability of inoculated spirochetes. Mice inoculated with > 10(4) live spirochetes and > 10(7) heat-killed spirochetes seroconverted to OspA, but mice inoculated with fewer spirochetes did not seroconvert to OspA at 2 weeks after inoculation. Growth temperature of spirochetes was not a factor for infectious dose or seroconversion to OspA. Spirochetes grown at 30, 34, or 38 degrees C had the same median infectious dose. Growth temperature did not influence infectious dose when mice were inoculated intraperitoneally or intradermally and did not influence dose-related immunologic recognition of OspA. Mice hyperimmunized with recombinant OspA-glutathione S-transferase (GT) fusion protein or GT (controls) were challenged by syringe inoculation with 10(3) spirochetes or by transplantation of infected skin from syngenic mice infected for 2 or 8 weeks. OspA-GT-immunized mice resisted syringe challenge but developed disseminated infections following transplantation of infected skin. Identical results were obtained in mice passively immunized with hyperimmune serum to OspA-GT or GT and then challenged by syringe or infected skin transplant. The number of spirochetes in infected skin, determined by quantitative PCR directed toward both plasmid and genomic targets, was less than the syringe challenge dose.     

Detection of Immune Complexes Is Not Independent of Detection of Antibodies in Lyme Disease Patients and Does Not Confirm Active Infection with Borrelia burgdorferi

The Borrelia burgdorferi-specific immune complex (IC) test, which uses polyethylene glycol (PEG) precipitation to isolate ICs from serum, has been used as a research test in the laboratory diagnosis of early Lyme disease (LD) and has been proposed as a marker of active infection. We examined whether B. burgdorferi-specific antibodies were present within PEG-precipitated ICs (PEG-ICs) in patients with LD, posttreatment Lyme disease syndrome, and controls, including individuals who received the outer surface protein A (OspA) vaccine. Using a B. burgdorferi whole-cell enzyme-linked immunosorbent assay (ELISA), we obtained positive PEG-IC results not only in patients with a history of LD, but also in individuals vaccinated with OspA vaccine. The frequency of positive PEG-IC ELISAs in OspA vaccinees was significantly higher with ELISA-reactive than with ELISA-negative unprocessed serum samples (P = 0.001), demonstrating dependency between the tests. Similar results were found using samples from rhesus macaques infected with B. burgdorferi, uninfected macaques vaccinated with OspA, and controls. Therefore, testing for the presence of antibodies against B. burgdorferi in PEG-IC preparations is not more likely to reflect active infection than testing in unprocessed serum and should not be used in individuals who received the OspA vaccine.     

Identification of Lysine Residues in the Borrelia burgdorferi DbpA Adhesin Required for Murine Infection

Decorin-binding protein A (DbpA) of Borrelia burgdorferi mediates bacterial adhesion to heparin and dermatan sulfate associated with decorin. Lysines K82, K163, and K170 of DbpA are known to be important for in vitro interaction with decorin, and the DbpA structure, initially solved by nuclear magnetic resonance (NMR) spectroscopy, suggests these lysine residues colocalize in a pocket near the C terminus of the protein. In the current study, we solved the structure of DbpA from B. burgdorferi strain 297 using X-ray crystallography and confirmed the existing NMR structural data. In vitro binding experiments confirmed that recombinant DbpA proteins with mutations in K82, K163, or K170 did not bind decorin, which was due to an inability to interact with dermatan sulfate. Most importantly, we determined that the in vitro binding defect observed upon mutation of K82, K163, or K170 in DbpA also led to a defect during infection. The infectivity of B. burgdorferi expressing individual dbpA lysine point mutants was assessed in mice challenged via needle inoculation. Murine infection studies showed that strains expressing dbpA with mutations in K82, K163, and K170 were significantly attenuated and could not be cultured from any tissue. Proper expression and cellular localization of the mutated DbpA proteins were examined, and NMR spectroscopy determined that the mutant DbpA proteins were structurally similar to wild-type DbpA. Taken together, these data showed that lysines K82, K163, and K170 potentiate the binding of DbpA to dermatan sulfate and that an interaction(s) mediated by these lysines is essential for B. burgdorferi murine infection.     

Specificity of Infection-Induced Immunity among Borrelia burgdorferi Sensu Lato Species

The specificity of infection-induced immunity in mice infected with cultured or host-adapted Borrelia burgdorferi sensu lato, the agent of Lyme disease, was examined. Sera obtained from mice following infection with high and low doses of cultured B. burgdorferi sensu stricto, transplantation of infected tissue (host-adapted spirochetes), or tick-borne inoculation all showed protective activity in passive immunization assays. Infection and disease were similar in mice infected with cultured spirochetes or by transplantation. Thus, the adaptive form of inoculated spirochetes did not influence the immune response during active infection. Mice infected with B. burgdorferi sensu stricto and then cured of infection with an antibiotic during early or late stages of infection were resistant to challenge with high doses of homologous cultured spirochetes for up to 1 year. In contrast, actively immune mice infected with different Borrelia species (B. burgdorferi sensu lato, B. burgdorferi sensu stricto cN40, Borrelia afzelii PKo, and Borrelia garinii PBi) and then treated with an antibiotic were resistant to challenge with cultured homologous but not heterologous spirochetes. Similar results were achieved for actively immune mice challenged by transplantation and by passive immunization with sera from mice infected with each of the Borrelia species and then challenged with cultured spirochetes. Arthritis and carditis in mice that had immunizing infections with B. afzelii and B. garinii and then challenged by transplantation with B. burgdorferi sensu stricto were equivalent in prevalence and severity to those in nonimmune recipient mice. These results indicate that protective immunity and disease-modulating immunity that develop during active infection are universal among species related to B. burgdorferi sensu lato but are species specific.     

Outer Surface Protein OspC Is an Antiphagocytic Factor That Protects Borrelia burgdorferi from Phagocytosis by Macrophages

Outer surface protein C (OspC) is one of the major lipoproteins expressed on the surface of Borrelia burgdorferi during tick feeding and the early phase of mammalian infection. OspC is required for B. burgdorferi to establish infection in both immunocompetent and SCID mice and has been proposed to facilitate evasion of innate immune defenses. However, the exact biological function of OspC remains elusive. In this study, we showed that the ospC-deficient spirochete could not establish infection in NOD-scid IL2rγ^null mice that lack B cells, T cells, NK cells, and lytic complement. The ospC mutant also could not establish infection in anti-Ly6G-treated SCID and C3H/HeN mice (depletion of neutrophils). However, depletion of mononuclear phagocytes at the skin site of inoculation in SCID and C3H/HeN mice allowed the ospC mutant to establish infection in vivo. In phagocyte-depleted mice, the ospC mutant was able to colonize the joints and triggered neutrophilia during dissemination. Furthermore, we found that phagocytosis of green fluorescent protein (GFP)-expressing ospC mutant spirochetes by murine peritoneal macrophages and human THP-1 macrophage-like cells, but not in PMN-HL60, was significantly higher than parental wild-type B. burgdorferi strains, suggesting that OspC has an antiphagocytic property. In addition, overproduction of OspC in spirochetes also decreased the uptake of spirochetes by murine peritoneal macrophages. Together, our findings provide evidence that mononuclear phagocytes play a key role in clearance of the ospC mutant and that OspC promotes spirochetes'' evasion of macrophages during early Lyme borreliosis.     

Bacterin That Induces Anti-OspA and Anti-OspC Borreliacidal Antibodies Provides a High Level of Protection against Canine Lyme Disease

Groups of 15 laboratory-bred beagles were vaccinated and boosted with either a placebo or adjuvanted bivalent bacterin comprised of a traditional Borrelia burgdorferi strain and a unique ospA- and ospB-negative B. burgdorferi strain that expressed high levels of OspC and then challenged with B. burgdorferi-infected Ixodes scapularis ticks. The vaccinated dogs produced high titers of anti-OspA and anti-OspC borreliacidal antibodies, including borreliacidal antibodies specific for an epitope within the last seven amino acids at the OspC carboxy terminus (termed OspC7) that was conserved among pathogenic Borrelia genospecies. In addition, spirochetes were eliminated from the infected ticks that fed on the bacterin recipients, B. burgdorferi was not isolated from the skin or joints, and antibody responses associated specifically with canine infection with B. burgdorferi were not produced. In contrast, B. burgdorferi was recovered from engorged ticks that fed on 13 (87%) placebo-vaccinated dogs (P < 0.0001), skin biopsy specimens from 14 (93%) dogs (P < 0.0001), and joint tissue specimens from 8 (53%) dogs (P = 0.0022). In addition, 14 (93%) dogs developed specific antibody responses against B. burgdorferi proteins, including 11 (73%) with C6 peptide antibodies (P < 0.0001). Moreover, 10 (67%) dogs developed Lyme disease-associated joint abnormalities (P < 0.0001), including 4 (27%) dogs that developed joint stiffness or lameness and 6 (40%) that developed chronic joint inflammation (synovitis). The results therefore confirmed that the bacterin provided a high level of protection against Lyme disease shortly after immunization.Dogs with Lyme disease rarely develop acute illness (26); but the infection reliably causes chronic subclinical polyarthritis and/or periarteritis (43) and occasionally causes frank recurrent arthritis with myalgia, fever, anorexia, and lethargy (41, 42); renal failure (11); heart block (24); or neurologic disease (9). In addition, the severity of the illness appears to be influenced by the species and the age of the dog. For example, beagle puppies are prone to oligoarthropathy (2, 39), while adults are more likely to develop asymptomatic synovitis (2, 7, 43). Moreover, Labrador retrievers, golden retrievers, and Shetland sheepdogs appear to be more susceptible to kidney nephropathy (11).Several commercial dog vaccines are currently available, and each provides protection primarily by inducing the production of anti-OspA borreliacidal antibodies that stimulate complement to form a membrane attack complex (33) that kills Borrelia burgdorferi in the tick midgut as the infected vectors ingest blood (12, 16). The approach has been effective (8, 10, 31, 40), but the vaccines may also fail (23) because the expression of OspA is downregulated immediately after the infected tick begins acquiring a blood meal (36), borreliacidal antibodies specific for OspA are genospecies specific (28, 49), and ticks can be infected with variant OspA-negative Lyme disease spirochetes (15).Another viable target for antibody-mediated immunity is OspC (18), especially since, in contrast to OspA, the Lyme disease spirochetes upregulate the expression of OspC as the tick begins feeding (36) and express OspC during the early stages of a mammalian infection (45). However, vaccines that provide protection by inducing anti-OspC antibodies have not been pursued aggressively, likely because the extreme heterogeneity, even among B. burgdorferi isolates from the same geographic area (44, 47), suggested that the protection afforded by anti-OspC antibodies would not be comprehensive.However, researchers (21) recently identified an epitope within the surface-exposed 7 amino acids of the carboxy terminus of OspC (hereafter referred to as OspC7) recognized by anti-OspC borreliacidal antibodies. More significantly, the epitope within the OspC7 region is conserved among the pathogenic Borrelia genospecies, including B. afzelii and B. garinii. Therefore, anti-OspC borreliacidal antibodies formed against the OspC7 region should provide comprehensive protection and should be effective against spirochetes in the tick and during the early stages of mammalian infection. Moreover, in contrast to the use of vaccination to induce anti-OspA borreliacidal antibodies, vaccination with proteins such as OspC that are also expressed during mammalian infection provides the possibility of producing an effective anamnestic immunologic memory response. We therefore developed a bivalent bacterin that induced both anti-OspA and anti-OspC borreliacidal antibodies, including borreliacidal antibodies specific for the conserved epitope within the OspC7 region, and evaluated the ability of immunization to provide protection against challenge from B. burgdorferi-infected ticks.(This study was presented in part at the 25th American College of Veterinary Internal Medicine Forum, Seattle, WA, 6 to 9 June 2007 [22a, 22b].)     

A Monoclonal Antibody Generated by Antigen Inoculation via Tick Bite Is Reactive to the Borrelia burgdorferi Rev Protein, a Member of the 2.9 Gene Family Locus

Murine monoclonal antibodies directed against proteins of Borrelia burgdorferi B31 (low passage) were generated by the administration of antigen via the bite of borrelia-infected ticks. This strategy was employed as a mechanism to create antibodies against antigens presented by the natural route of tick transmission versus those presented by inoculation with cultured borreliae. One of the resultant antibodies reacted with a 17-kDa antigen from cultured B. burgdorferi, as seen by immunoblot analysis. This antibody was used to screen a B. burgdorferi genomic DNA lambda vector expression library, and an immunoreactive clone was isolated. DNA sequence analysis of this clone, containing a 2.7-kb insert, revealed several open reading frames. These open reading frames were found to be homologs of genes discovered as a multicopy gene family in the 297 strain of B. burgdorferi by Porcella et al. (S. F. Porcella, T. G. Popova, D. R. Akins, M. Li, J. D. Radolf, and M. V. Norgard, J. Bacteriol. 178:3293–3307, 1996). By selectively subcloning genes found in this insert into an Escherichia coli plasmid expression vector, the observation was made that the rev gene product was the protein reactive with the 17-kDa-specific monoclonal antibody. The rev gene product was found to be expressed in low-passage, but not in high-passage, B. burgdorferi B31. Correspondingly, the rev gene was not present in strain B31 genomic DNA from cultures that had been passaged >50 times. Serum samples from Lyme disease patients demonstrated an antibody response against the Rev protein. The generation of an anti-Rev response in Lyme disease patients, and in mice by tick bite inoculation, provides evidence that the Rev protein is expressed and immunogenic during the course of natural transmission and infection.     

Evaluation of RevA,a Fibronectin-Binding Protein of Borrelia burgdorferi,as a Potential Vaccine Candidate for Lyme Disease

Previous studies indicated that the Lyme disease spirochete Borrelia burgdorferi expresses the RevA outer surface protein during mammalian infection. As an adhesin that promotes bacterial interaction with fibronectin, RevA appears to be a good target for preventive therapies. RevA proteins are highly conserved across all Lyme borreliae, and antibodies against RevA protein are cross-reactive among RevA proteins from diverse strains. Mice infected with B. burgdorferi mounted a rapid IgM response to RevA, followed by a strong IgG response that generally remained elevated for more than 12 months, suggesting continued exposure of RevA protein to the immune system. RevA antibodies were bactericidal in vitro. To evaluate the RevA antigen as a potential vaccine, mice were vaccinated with recombinant RevA and challenged with B. burgdorferi by inoculation with a needle or by a tick bite. Cultured tissues from all treatment groups were positive for B. burgdorferi. Vaccinated animals also appeared to have similar levels of B. burgdorferi DNA compared to nonvaccinated controls. Despite its antigenicity, surface expression, and the production of bactericidal antibodies against it, RevA does not protect against Borrelia burgdorferi infection in a mouse model. However, passive immunization with anti-RevA antibodies did prevent infection, suggesting the possible utility of RevA-based immunotherapeutics or vaccine.     

Roles of OspA, OspB, and flagellin in protective immunity to Lyme borreliosis in laboratory mice.

Vaccination with recombinant outer surface protein A (OspA) has been shown to protect mice from infection with Borrelia burgdorferi, the Lyme disease agent. To determine whether antibodies to B. burgdorferi proteins other than OspA are involved in protective immunity, antibodies to OspA were removed from protective anti-B. burgdorferi serum; the residual serum was still protective. Absorption of OspA and OspB antibodies from anti-B. burgdorferi serum eliminated the protective effect. Therefore, active immunization experiments were performed to determine the roles of OspB and flagellin in protective immunity and to determine whether protective immunity induced by OspA is dose dependent. Active immunization with recombinant OspA protected mice from infection with an inoculum of 10(4) spirochetes, but this protection could be overcome with a challenge of 10(7) spirochetes; OspB protected mice from infection with an inoculum of 10(3) spirochetes but was insufficient to fully protect against 10(4) organisms; and immunization with flagellin had no protective effect. These studies suggest that OspA and OspB, but not flagellin, play roles in protective immunity to spirochete infection.